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TRK-fused gene (TFG) regulates ULK1 stability via TRAF3-mediated ubiquitination and protects macrophages from LPS-induced pyroptosis.

ABSTRACT: TRK-fused gene (TFG) is known to be involved in protein secretion and plays essential roles in an antiviral innate immune response. However, its function in LPS-induced inflammation and pyroptotic cell death is still unknown. Here, we reported that TFG promotes the stabilization of Unc-51 like autophagy activating kinase (ULK1) and participates in LPS plus nigericin (Ng) induced pyroptotic cell death. Our results showed that TFG-deficient THP-1 macrophages exhibit higher mitochondrial ROS production. LPS/Ng stimulation triggers a much higher level of ROS and induces pyroptotic cell death. ULK1 undergoes a rapid turnover in TFG-deficient THP-1 cells. TFG forms complex with an E3 ligase, tumor necrosis factor receptor-associated factor 3 (TRAF3), and stabilizes ULK1 via disturbing ULK1-TRAF3 interaction. Knockdown of TFG facilitates the interaction of ULK1 with TRAF3 and subsequent K48-linked ULK1 ubiquitination and proteasome degradation. Rescue of ULK1 expression blocks LPS/Ng-induced cell death in TFG-deficient THP-1 macrophages. Taken together, TFG plays an essential role in LPS/Ng-induced pyroptotic cell death via regulating K48-linked ULK1 ubiquitination in macrophages.

SUBMITTER: Shi JH

PROVIDER: S-EPMC8795729 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and aimsThe immune-inflammatory cascade and pyroptosis play an important role in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). The maintenance of immune homeostasis is inextricably linked to the Notch signaling pathway, but whether myeloid Notch1 affects microglia polarization as well as neuronal pyroptosis in CIRI is not fully understood. This study was designed to clarify the role of myeloid Notch1 in CIRI, providing new therapeutic strategies for ischemic stroke.Methods and resultsMyeloid-specific Notch1 knockout (Notch1M-KO) mice and the floxed Notch1 (Notch1FL/FL) mice were subjected to middle cerebral artery occlusion (MCAO). After 3 days of CIRI, we evaluated the neurological deficit score and cerebral infarction volume. Immunofluorescence staining was used to detect the expression of Notch1 and microglial subtype markers. Cerebral infiltrating macrophages were detected by flow cytometry. RT-qPCR was used to detect pro-inflammatory cytokines. Western blot was used to detect the expression of pyroptosis related proteins. The Notch1-siRNA transfected BV2 cells were co-cultured with HT22 cells to investigate the potential mechanisms by which microglial Notch1 affects neuronal pyroptosis induced by anoxia/reoxygenation in vitro. We found that Notch1 was activated in cerebral microglia/macrophages after CIRI. Myeloid Notch1 deficiency decreased the cerebral infarct volume (24.17 ± 3.29 vs. 36.17 ± 2.27, p < 0.001), neurological function scores (2.33 ± 0.47 vs. 3.17 ± 0.37, p < 0.001) and the infiltration of peripheral monocytes/macrophages (3.26 ± 0.53 vs. 5.67 ± 0.57, p < 0.01). Strikingly, myeloid-specific Notch1 knockout alleviated pyroptosis. Compared with microglia M1, increased microglia M2 were detected in the ischemic penumbra. In parallel in vitro co-culture experiments, we found that Notch1 knockdown in microglial BV2 cells inhibited anoxia/reoxygenation-induced JAK2/STAT3 activation and pyroptosis in hippocampal neuron HT22 cells.ConclusionsOur findings elucidate the underlying mechanism of the myeloid Notch1 signaling pathway in regulating neuronal pyroptosis in CIRI, suggesting that targeting myeloid-specific Notch1 is an effective strategy for the treatment of ischemic stroke.

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TRK-fused gene (TFG) regulates ULK1 stability via TRAF3-mediated ubiquitination and protects macrophages from LPS-induced pyroptosis.

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