Abstract
Background
Necroptosis, a form of programmed necrosis, has been shown to contribute to the pathogenesis of various diseases including ischemia/reperfusion injury and heart failure. We recently reported that necroptotic signals suppresses autophagy in cardiomyocytes and that rapamycin, an mTORC1 inhibitor, not only promotes autophagy but also protect the cells from necroptosis.
Purpose
We examined the mechanism by which rapamycin suppresses necroptosis of cardiomyocytes, focusing on regulation of RIP1 activity and autophagic flux.
Methods and results
In H9c2 cardiomyoblasts, necroptosis was induced by treatment with TNF and z-VAD-fmk (zVAD) for 24 h, and cell death was determined by LDH release (as % of total). The treatment with TNF/zVAD increased LDH release from 3.4±1.3% to 46.1±2.3%, and LDH release was suppressed by necrostatin-1 (5.9±0.9%), a RIP1 inhibitor, and by rapamycin (23.5±1.4%). The protective effect of rapamycin was mimicked by Ku-0063794, an mTORC1/2 inhibitor. TNF/zVAD induced RIP1-RIP3 complex formation, together with suppression of TNF-induced RIP1 cleavage, which was mitigated by rapamycin. In addition, rapamycin not only suppressed TNF/zVAD-induced phosphorylation of RIP1-Ser166, an index of RIP1 activation, but also increased phosphorylation of RIP1-Ser320, an inhibitory phosphorylation site. In cells transfected with RIP1-S320A, which lack Ser320 for inhibitory phosphorylation, rapamycin failed to suppress TNF/zVAD-induced RIP1-RIP3 binding and cell death. Immunoblot analyses showed that TNF/zVAD significantly increased level of LC3-II. The accumulation of LC3-II protein was not further increased by bafilomycin A1 (100 nM), an inhibitor of lysosomal protein degradation, indicating that accumulation of LC3-II by TNF/zVAD reflected suppression of autophagic flux. Inhibition of RIP1 by necrostatin-1 attenuated TNF/zVAD-induced accumulation of LC3 II. The restoration of autophagic flux in TNF/zVAD-treated cells by necrostatin-1 was confirmed by monitoring tandem RFP-GFP-LC3 transfected cells; necrostatin-1 increased a ratio of RFP-LC3-puncta (autolysosomes) to RFP-GFP-LC3-puncta (autophagosomes) in TNF/zVAD-treated cells. In addition, necrostatin-1 and rapamycin induced nuclear translocation of TFEB, a regulator of lysosome biogenesis, which was associated with upregulation of MCOLN1 mRNA, a downstream target of TFEB. Restoration of autophagic flux in TNF/zVAD-treated cells by necrostatin-1 was inhibited by siRNA-mediated knockdown of TFEB.
Conclusion
Activation of TFEB by inhibitiory phosphorylation of RIP1-Ser320 is a primary mechanism of cytoprotection afforded by mTORC1 inhibition against necroptosis.
Multiple Parameters Beyond Lipid Binding Affinity Drive Cytotoxicity of Cholesterol-Dependent Cytolysins
