Background/Purpose: In our previous study, we discovered that ursolic acid (UA), a pentacyclic triterpenoid with anti-inflammatory properties, induces apoptosis in synovial fibroblasts from rheumatoid arthritis patients (RASFs) by upregulating Noxa and downregulating Mcl-1. To examine the molecular mechanism of UA-mediated apoptosis, we tested early signaling pathways that may activate Noxa expression as well as potential mechanisms of Noxa-mediated proteasomal degradation of Mcl-1 in human RASFs.

Methods: Human RASFs were treated with UA (5-10 μM) to determine the activated signaling pathways by Western blotting and qRT-PCR methods. The spatial association of Noxa with Mcl-1 and other E3 ligases was studied using immunoprecipitation (IP), immunofluorescence (IF), and proximal ligation assay (PLA) methods. The ubiquitination of Mcl-1 was studied using denatured IP.

Results: Treatment of RASFs with UA (5-10 μM) resulted in a time- and dose-dependent increase in Noxa, but decrease in Mcl-1 expression. Addition of proteasome inhibitor (MG132) showed no further increase in Noxa expression, suggesting transcriptional activation as a potential mechanism behind UA-induced Noxa expression. Evaluation of the signaling pathways showed that UA induced temporal expression of hypoxia-inducible factor-1 α (HIF-1α) in RASFs. Furthermore, our results showed that UA utilizes Akt, p38-MAPK, and JNK/SAPK signaling pathways to activate Noxa expression in RASFs. Utilizing inhibitors of JNK/SAPK (SP600125), p38-MAPK (SB203580), Akt (LY294002), and HIF-1a (HIF-1a inhibitor) pathways, we showed that inhibition of JNK/SAPK pathway significantly reduced UA-mediated upregulation of Noxa. In addition, the results from PLA and IF studies in UA-treated RASFs showed that Noxa co-localizes with Mcl-1 predominantly in the non-nuclear compartment of the cell, suggesting its association with Mcl-1 in ‘priming’ Mcl-1 for proteasomal degradation. Interestingly, the IP of Mcl-1 under denatured condition after UA treatment and subsequent Western blot analysis showed an increase in K48-linked, but not K63-linked, ubiquitin chains tagged to Mcl-1. This K48 polyubiquitylation of Mcl-1 appears to be mediated by Mule, a HECT domain containing E3 ubiquitin-protein ligase, since UA treatment of RASFs preferentially induced the recruitment of Mule rather than SCF^β-TrCP to Noxa/Mcl-1 complex to facilitate Mcl-1 degradation.

Conclusion: Taken together, these results suggest that UA induces Noxa expression via JNK pathway and facilitates Mcl-1 association with E3 ubiquitin ligase Mule to activate Mcl-1 degradation that sensitizes RASFs to apoptosis. These findings not only unveil a novel mechanism of UA in inducing apoptosis of RAFSs, but also a potential new approach to treat RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ursolic-acid-promotes-apoptosis-of-rheumatoid-athritis-synovial-fibroblasts-by-upregulating-noxa-expression-and-recruiting-e3-ligase-mule-to-degrade-mcl-1/