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TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy.

ABSTRACT: Macroautophagy/autophagy and necroptosis represent two opposing cellular s tress responses. Whereas autophagy primarily fulfills a cyto-protective function, necroptosis is a form of regulated cell death induced via death receptors. Here, we aimed at investigating the molecular crosstalk between these two pathways. We observed that RIPK3 directly associates with AMPK and phosphorylates its catalytic subunit PRKAA1/2 at T183/T172. Activated AMPK then phosphorylates the autophagy-regulating proteins ULK1 and BECN1. However, the lysosomal degradation of autophagosomes is blocked by TNF-induced necroptosis. Specifically, we observed dysregulated SNARE complexes upon TNF treatment; e.g., reduced levels of full-length STX17. In summary, we identified RIPK3 as an AMPK-activating kinase and thus a direct link between autophagy- and necroptosis-regulating kinases.Abbreviations: ACACA/ACC: acetyl-CoA carboxylase alpha; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; GFP: green fluorescent protein; EBSS: Earle's balanced salt solution; Hs: Homo sapiens; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain like pseudokinase; Mm: Mus musculus; MTOR: mechanistic target of rapamycin kinase; MVB: multivesicular body; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PLA: proximity ligation assay; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2: protein kinase AMP-activated catalytic subunit alpha 2; PRKAB2: protein kinase AMP-activated non-catalytic subunit beta 2; PRKAG1: protein kinase AMP-activated non-catalytic subunit gamma 1; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; STX7: syntaxin 7; STX17: syntaxin 17; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

SUBMITTER: Wu W

PROVIDER: S-EPMC8726653 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cilia are sensory organelles that project from the surface of almost all cells. Nephronophthisis (NPH) and NPH-related ciliopathies are degenerative genetic diseases caused by mutation of cilia-associated genes. These kidney disorders are characterized by progressive loss of functional tubular epithelial cells which is associated with inflammation, progressive fibrosis, and cyst formation, ultimately leading to end-stage renal disease. However, disease mechanisms remain poorly understood. Here, we show that targeted deletion of cilia in renal epithelial cells enhanced susceptibility to necroptotic cell death under inflammatory conditions. Treatment of non-ciliated cells with tumor necrosis factor (TNF) α and the SMAC mimetic birinapant resulted in Ripk1-dependent cell death, while viability of ciliated cells was almost not affected. Cell death could be enhanced and shifted toward necroptosis by the caspase inhibitor emricasan, which could be blocked by inhibitors of Ripk1 and Ripk3. Moreover, combined treatment of ciliated and non-ciliated cells with TNFα and cycloheximide induced a cell death response that could be partially rescued with emricasan in ciliated cells. In contrast, non-ciliated cells responded with pronounced cell death that was blocked by necroptosis inhibitors. Consistently, combined treatment with interferon-γ and emricasan induced cell death only in non-ciliated cells. Mechanistically, enhanced necroptosis induced by loss of cilia could be explained by induction of Ripk3 and increased abundance of autophagy components, including p62 and LC3 associated with the Ripk1/Ripk3 necrosome. Genetic ablation of cilia in renal tubular epithelial cells in mice resulted in TUNEL positivity and increased expression of Ripk3 in kidney tissue. Moreover, loss of Nphp1, the most frequent cause of NPH, further increased susceptibility to necroptosis in non-ciliated epithelial cells, suggesting that necroptosis might contribute to the pathogenesis of the disease. Together, these data provide a link between cilia-related signaling and cell death responses and shed new light on the disease pathogenesis of NPH-related ciliopathies.

Project description:BackgroundNotch signaling is highly conserved and critically involved in cell differentiation, immunity, and survival. Activation of the Notch pathway modulates immune cell functions during the inflammatory response. However, it remains unknown whether and how the macrophage Notch1 may control the innate immune signaling TAK1, and RIPK3-mediated hepatocyte necroptosis in liver ischemia and reperfusion injury (IRI). This study investigated the molecular mechanisms of macrophage Notch1 in modulating TAK1-mediated innate immune responses and RIPK3 functions in liver IRI.MethodsMyeloid-specific Notch1 knockout (Notch1M-KO) and floxed Notch1 (Notch1FL/FL) mice (n = 6/group) were subjected to 90 min partial liver warm ischemia followed by 6 h of reperfusion. In a parallel in vitro study, bone marrow-derived macrophages (BMMs) were isolated from these conditional knockout mice and transfected with CRISPR/Cas9-mediated β-catenin knockout (KO) vector followed by LPS (100 ng/ml) stimulation.ResultsIR stress-induced Notch1 activation evidenced by increased nuclear Notch intracellular domain (NICD) expression in liver macrophages. Myeloid Notch1 deficiency exacerbated IR-induced liver damage, with increased serum ALT levels, macrophage/neutrophil accumulation, and proinflammatory cytokines/chemokines production compared to the Notch1FL/FL controls. Unlike in the Notch1FL/FL controls, Notch1M-KO enhanced TRAF6, TAK1, NF-κB, RIPK3, and MLKL but reduced β-catenin activation in ischemic livers. However, adoptive transfer of lentivirus β-catenin-modified macrophages markedly improved liver function with reduced TRAF6, p-TAK1, RIPK3 and p-MLKL in IR-challenged livers. Moreover, disruption of RIPK3 in Notch1M-KO mice with an in vivo mannose-mediated RIPK3 siRNA delivery system diminished IR-triggered hepatocyte death. In vitro studies showed that macrophage NICD and β-catenin co-localized in the nucleus, whereby β-catenin interacted with NICD in response to LPS stimulation. Disruption of β-catenin with a CRISPR/Cas9-mediated β-catenin KO in Notch1FL/FL macrophage augmented TRAF6 activation leading to enhanced TAK1 function. While CRISPR/Cas9-mediated TRAF6 KO in Notch1M-KO macrophage inhibited RIPK3-mediated hepatocyte necroptosis after co-culture with primary hepatocytes.ConclusionsMacrophage Notch1 controls TAK1-mediated innate immune responses and RIPK3-mediated hepatocyte necroptosis through activation of β-catenin. β-catenin is required for the macrophage Notch1-mediated immune regulation in liver IRI. Our findings demonstrate that the macrophage Notch1-β-catenin axis is a crucial regulatory mechanism in IR-triggered liver inflammation and provide novel therapeutic potential in organ IRI and transplant recipients. Video abstract.

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TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy.

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