Novel targeted therapeutics for regulating synovial hyperplasia in RA



 DESCRIPTION (provided by applicant): In rheumatoid arthritis (RA), an increased expression of anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1), a Bcl-2 family member, contributes to synovial hyperplasia and resistance of RA fibroblast-like synoviocytes (RA- FLS) to apoptosis. Mcl-1 mediated resistance to apoptosis in RA-FLS may partly be attributed to the lack in expression of endogenous pro-apoptotic Bcl-2 homology 3 (BH3)-only proteins that can bind Mcl-1 and others. Among the BH3-only family, Noxa stands out exceptional for its specificity to bind Mcl-1. Noxa's binding to Mcl-1 facilitates structural modification that allows binding of Mcl- Ubiquitin ligase E3 (Mule) to ubiquitinate and `prime' Mcl-1 for proteasomal degradation. Pharmacological strategies aimed at enhancing Noxa expression or delivering BH3 mimetics, which are yet to be tested in RA, may have significant therapeutic impact in regulating synovial hyperplasia in RA. In this regard have identified a pentacyclic triterpenoid, ursolic acid (UA), as a potent inducer of Noxa expression in human RA-FLS. Comparative mRNA analysis showed that Mcl-1/Noxa ratio was >2-fold higher in RA-FLS compared to normal human FLS (NL-FLS). Interestingly, UA treatment flipped this ratio by inducing Noxa expression and selectively inhibiting Mcl-1, not Bcl-2, expression to induce RA-FLS apoptosis. U Bcl-2 proteins to blunt their biological activities sing WT and Noxa-/- BMK mouse kidney cell line, we found that UA was ineffective in inducing apoptosis in Noxa-/- cells. Interestingly, transfection of Noxa-/- BMK cells with stably expressing Noxa restored the apoptosis inducing capabilities of UA. A marked increase in the expression of polyubiqitinated proteins in RA-FLS was also observed compared to the NL-FLS upon UA treatment. In addition, UA-induced Mcl-1 degradation was inhibited by proteasome inhibitor (MG132) validating the role of ubiquitin-proteasome system (UPS) in RA-FLS apoptosis. UA-induced Noxa expression. This proposal capitalizes on these novel observations, and the central hypothesis of our grant is that UA suppresses synovial hyperplasia and sensitizes RA-FLS to apoptosis by inducing Noxa expression. Studies proposed in Aim 1 will determine the molecular mechanisms involved in UA-induced Noxa upregulation and activation of UPS to `prime' Mcl-1 for proteasomal degradation and RA-FLS apoptosis. In Aim 2, we will characterize and compare phosphorylation sites on Mcl-1 in FLS from NL, OA, or RA donors and study the effect of UA in modulating them to initiate Mcl-1 degradation and sensitize RA-FLS to apoptosis. The success of these studies will provide the molecular mechanisms of Noxa-induced Mcl- 1 degradation and the therapeutic importance of BH3-only protein inducers such as UA or its structural analogs in regulating RA synovial hyperplasia.