MLKL and CaMKII Are Involved in RIPK3-Mediated Smooth Muscle Cell Necroptosis.

Bo Liu,Ting Zhou,Amelia Stranz,Elise Deroo,Roman Ginnan,Qiwei Wang,Huan Yang,Harold A Singer

doi:10.3390/cells10092397

Abstract

Receptor interacting protein kinase 3 (RIPK3)-mediated smooth muscle cell (SMC) necroptosis has been shown to contribute to the pathogenesis of abdominal aortic aneurysms (AAAs). However, the signaling steps downstream from RIPK3 during SMC necroptosis remain unknown. In this study, the roles of mixed lineage kinase domain-like pseudokinase (MLKL) and calcium/calmodulin-dependent protein kinase II (CaMKII) in SMC necroptosis were investigated. We found that both MLKL and CaMKII were phosphorylated in SMCs in a murine CaCl2-driven model of AAA and that Ripk3 deficiency reduced the phosphorylation of MLKL and CaMKII. In vitro, mouse aortic SMCs were treated with tumor necrosis factor α (TNFα) plus Z-VAD-FMK (zVAD) to induce necroptosis. Our data showed that both MLKL and CaMKII were phosphorylated after TNFα plus zVAD treatment in a time-dependent manner. SiRNA silencing of Mlkl-diminished cell death and administration of the CaMKII inhibitor myristoylated autocamtide-2-related inhibitory peptide (Myr-AIP) or siRNAs against Camk2d partially inhibited necroptosis. Moreover, knocking down Mlkl decreased CaMKII phosphorylation, but silencing Camk2d did not affect phosphorylation, oligomerization, or trafficking of MLKL. Together, our results indicate that both MLKL and CaMKII are involved in RIPK3-mediated SMC necroptosis, and that MLKL is likely upstream of CaMKII in this process.

Highlights

Smooth muscle cell (SMC) death plays a significant role in the pathophysiology of abdominal aortic aneurysm (AAA) [1], a relatively common aortic disease characterized by progressive expansion and in some cases life-threatening rupture of the abdominal aorta
Our lab has previously shown that receptor interacting protein kinase 3 (RIPK3)mediated SMC necroptosis contributes to AAA pathology [2]
We found that both mixed lineage kinase domain-like pseudokinase (MLKL) and calmodulindependent protein kinase II (CaMKII) were phosphorylated in aortic tissue from mice with CaCl2 -induced AAAs and in aortic SMC undergoing necroptosis after tumor necrosis factor α (TNFα) plus Z-VAD-FMK treatment

Summary

Introduction

Smooth muscle cell (SMC) death plays a significant role in the pathophysiology of abdominal aortic aneurysm (AAA) [1], a relatively common aortic disease characterized by progressive expansion and in some cases life-threatening rupture of the abdominal aorta. Our lab has previously shown that receptor interacting protein kinase 3 (RIPK3)mediated SMC necroptosis contributes to AAA pathology [2]. Necroptosis is a programmed form of necrotic cell death distinct from necrosis and apoptosis. In the setting of inflammatory stimuli, RIPK3 and its signaling partner receptor interacting protein kinase. 1 (RIPK1) become phosphorylated and lead to necroptosis [3]. Our lab has demonstrated that aortic tissue from AAA patients have elevated levels of RIPK3 [2]. Inhibition of necroptosis either by gene deletion of Ripk or by chemical inhibitors to RIPK1 or both

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