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Updated: Jan 19, 2026
Feedback Inhibition of Enzymes
mTORC1 inhibition attenuates necroptosis through RIP1 inhibition-mediated TFEB activation
Koki Abe1, Toshiyuki Yano1, Masaya Tanno1
1Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Abstract:
Accumulating evidence indicates that necroptosis contributes to cardiovascular diseases. We recently reported suppression of autophagy by necroptotic signals in cardiomyocytes and protective action of rapamycin. Here we examined the mechanism by which mTORC1 inhibition protects cardiomyocytes from necroptosis. Necroptosis of H9c2 cells was induced by treatment with tumor necrotic factor-α (TNF) and z-VAD-fmk (zVAD), and the extent of necroptosis was determined as the level of LDH release (as % of total). TNF/zVAD increased RIP1-RIP3 interaction and LDH release from 3.4 ± 1.3% to 46.1 ± 2.3%. The effects of TNF/zVAD were suppressed by an mTORC1 inhibitor, rapamycin, and an mTORC1/2 inhibitor, Ku-0063794, but not by a p70s6K inhibitor, PF-4708671. Protection by rapamycin was not abolished by inhibitors of TAK1, IKKα/β, and cIAP, endogenous necroptosis suppressors upstream of RIP1. Rapamycin and Ku-0063794 suppressed TNF/zVAD-induced RIP1-Ser166 phosphorylation and increased phosphorylation of RIP1-Ser320, an inhibitory phosphorylation site, though such an effect on RIP1-Ser320 was not observed for PF-4708671. Protective effects of rapamycin on TNF/zVAD-induced RIP1-RIP3 binding and necroptosis were undetected in cells transfected with RIP1-S320A. In TNF/zVAD-treated cells, rapamycin and a RIP1 inhibitor, necrostatin-1, increased nuclear localization of transcriptional factor EB (TFEB) and promoted autolysosome formation from autophagosomes in a TFEB-dependent manner. Knockdown of TFEB expression attenuated rapamycin-induced protection from necroptosis in TNF/zVAD-treated cells. The results suggest that mTORC1 inhibition promotes autophagy and protects cardiomyocytes from necroptosis by a TFEB-dependent mechanism and that inhibition of RIP1 by increased phosphorylation at Ser320 is crucial in the cardiomyocyte protection afforded by mTORC1 inhibition.
Insights
mTORC1 inhibition protects cardiomyocytes from necroptosis by promoting autophagy via TFEB. This process involves inhibiting RIP1 through phosphorylation at Ser320, crucial for cell survival in cardiovascular disease contexts.
Area of Science:
- Cardiovascular Biology
- Cell Death Pathways
- Molecular Medicine
Background:
- Necroptosis, a programmed form of necrosis, is implicated in cardiovascular diseases.
- Necroptotic signals can suppress autophagy in cardiomyocytes, a process potentially counteracted by rapamycin.
- Understanding the protective mechanisms of mTORC1 inhibition against necroptosis is crucial for cardiovascular health.
Purpose of the Study:
- To elucidate the molecular mechanisms by which mTORC1 inhibition protects cardiomyocytes from necroptosis.
- To investigate the role of RIP1 phosphorylation and TFEB activation in this protective pathway.
- To explore the therapeutic potential of targeting mTORC1 in necroptosis-related cardiovascular conditions.
Main Methods:
- Induction of necroptosis in H9c2 cells using tumor necrosis factor-α (TNF) and z-VAD-fmk (zVAD).
- Assessment of necroptosis via lactate dehydrogenase (LDH) release and RIP1-RIP3 interaction.
- Pharmacological inhibition of mTORC1 and related kinases, alongside analysis of RIP1 phosphorylation at Ser166 and Ser320.
- Evaluation of TFEB nuclear localization, autophagosome-lysosome fusion, and the impact of TFEB knockdown.
Main Results:
- TNF/zVAD treatment induced significant necroptosis, characterized by increased RIP1-RIP3 interaction and LDH release.
- mTORC1 inhibitors (rapamycin, Ku-0063794) suppressed TNF/zVAD-induced necroptosis and RIP1-RIP3 binding.
- Protection was linked to suppressed RIP1-Ser166 phosphorylation and enhanced RIP1-Ser320 phosphorylation, with RIP1-S320A mutation abolishing protection.
- mTORC1 inhibition promoted TFEB nuclear translocation and autolysosome formation in a TFEB-dependent manner, attenuating necroptosis.
Conclusions:
- mTORC1 inhibition confers cardioprotection against necroptosis through a TFEB-dependent autophagy-promoting mechanism.
- Inhibition of RIP1 via increased Ser320 phosphorylation is a critical step in mTORC1-mediated cardioprotection.
- Targeting the mTORC1-TFEB-autophagy axis offers a potential therapeutic strategy for necroptosis-related cardiovascular diseases.
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