Background/Purpose:  In rheumatoid arthritis (RA), the paucity of pro-apoptotic protein expression may significantly contribute to the resistance of synovial fibroblasts (FLS) to apoptosis. In the present study, we evaluated if inducing the expression of pro-apoptotic protein Noxa in RA-FLS using a potent anti-inflammatory pentacyclic triterpenoid uroslic acid (UA) triggers apoptosis and studied the underlying mechanism.

Methods:  Effects of UA (2.5-10 μM) on human RA-FLS morphology and cell viability were determined by microscopy and a colorimetric MTT/SRB cell viability assays. Mechanism of UA’s activity was modulated experimentally by using the siRNA or plasmid overexpression approaches. Epigenetic regulation of Noxa by microRNA-181a (mir-181a) was studied using the microarray and qRT-PCR methods. Apoptosis was measured by the cleavage of caspase-3 and poly-ADP-ribose polymerase (PARP). Western blotting was used to evaluate the apoptosis signaling mediators, Noxa, and Mcl-1 expression. 

Results:  UA (2.5-20 μM) decreased the cell viability of RA-FLS in a dose-dependent manner. Importantly, UA (10 μM) selectively induced Noxa expression within 3 h to ~2-3 fold in RA-FLS (p<0.05; n=4). Induction of Noxa led to the consequent downregulation of Mcl-1 expression and apoptosis by 24 h of UA treatment (p<0.05; n=3). The inhibition of Mcl-1 expression by UA resulted in the sensitization of RA-FLS to TRAIL-induced PARP cleavage and apoptosis. Overexpression of Noxa using a plasmid vector targeting Noxa was effective in making RA-FLS susceptible to apoptosis. Using a siRNA method to block Noxa expression, we found that the RA-FLS apoptosis-inducing activity of UA was significantly blocked suggesting that UA induced apoptosis in RA-FLS primarily through Noxa upregulation. Confirmatory studies using WT and Noxa-/- BMK cells showed that UA efficiently induced apoptosis in WT cells but had no effect in Noxa-/- counterparts (p<0.01; two independent experiments). Interestingly, transfection of stably expressing Noxa in Noxa-/- BMK cells restored the apoptosis inducing capability of UA. MicroRNA array analysis showed a significant decrease in RA-FLS mir-181a expression, a miRNA known to facilitate apoptosis, by ~30% as compared to the normal FLS (p<0.05). We also found that UA (5-10 μM) was capable of inducing mir-181a expression in RA-FLS as compared to the non-stimulated samples suggesting that UA-induced Noxa expression and consequent apoptosis in RA-FLS may be mediated epigenetically via upregulation of mir-181a.

Conclusion:  Our novel findings indicate that inducing Noxa expression by UA in RA-FLS effectively induces apoptosis and this effect is partly mediated through mir-181a. Thus, developing therapeutic strategies that can selectively upregulate Noxa  and/or modulate mir-181a to induce apoptosis in RA-FLS may have potential therapeutic application for the treatment of RA.

---

« Back to 2014 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/induction-of-pro-apoptotic-noxa-expression-by-ursolic-acid-sensitizes-rheumatoid-arthritis-synovial-fibroblasts-to-apoptosis-a-role-of-mir-181a/