Proteasome inhibitor PS-341 limits macrophage necroptosis by promoting cIAPs-mediated inhibition of RIP1 and RIP3 activation

Abstract

Apoptotic and necrotic macrophages have long been known for their existence in atherosclerotic lesions. However, the mechanisms underlying the choice of their death pattern have not been fully elucidated. Here, we report the effects of PS-341, a potent and specific proteasome inhibitor, on the cell death of primary bone marrow-derived macrophages (BMDMs) in vitro. The results showed that PS-341 could not induce macrophage apoptosis or promote TNF-induced macrophage apoptosis, on the other hand, PS-341 could significantly inhibit macrophage necroptosis induced by TNF and pan-caspase inhibitor z-VAD treatment. Remarkably, high-dose of PS-341 showed similar inhibitory effects on macrophage necroptosis comparable to that of kinase inhibition of RIP1 through specific inhibitor Nec-1 or inhibition of RIP3 via specific genetical ablation. Furthermore, the degradation of cellular inhibitor of apoptosis proteins (cIAPs) was suppressed by PS-341, which could antagonize the activation of RIP1 kinase via post-translational mechanism. Further evidences demonstrated reduced levels of both RIP1 and RIP 3 upon PS-341 treatment, concomitantly, a more strong association of RIP1 with cIAPs and less with RIP3 was found following PS-341 treatment, these findings suggested that PS-341 may disrupt the formation of RIP1-RIP3 complex (necrosome) through stabilizing cIAPs. Collectively, our results indicated that the proteasome-mediated degradation of cIAPs could be inhibited by PS-341 and followed by limited RIP1 and RIP3 kinase activities, which were indispensable for necroptosis, thus eliciting a significant necroptosis rescue in BMDMs in vitro. Overall, our study has identified a new role of PS-341 in the cell death of BMDMs and provided a novel insight into the atherosclerotic inflammation caused by proteasome-mediated macrophage necroptosis.