CD9-dependent extracellular vesicles mediate the propagation of ferroptosis. CD9-dependent extracellular vesicles mediate the propagation of ferroptosis

Ferroptosis is a form of regulated cell death that is mediated by accumulation of lipid peroxidation. Emerging evidences report dispersion of ferroptosis between neighboring cells. However, the molecular mechanism underlying the propagation of ferroptosis remains unclear. Here we provide evidences that ferroptotic cells elicit cellular projections that produce massive extracellular vesicles (EVs). Proteomic analysis reveals that these EVs are CD9 positive and contain major components of mitogen-activated protein kinase (MAPK) signaling. MAP2K1/2 that is transferred by EVs increases the sensitivity to ferroptosis of target cells. We further show that a MAP2K-interacting transcription factor, peroxisome-proliferator activator receptor gamma (PPARG), activates various components of ferroptosis defense systems. Moreover, perturbation of EV-based transport protects adjacent tissue from ferroptosis in vivo. Our study reveals an EV-dependent transport between ferroptotic cells and surrounding neighbors and establishes that MAPK-responsive PPARG is essential for ferroptosis defense in recipient cells. Overall design: U-2 OS cells expressing PPARG-EGFP were used CUT&Tag-sequencing to analyze PPARG regulating genes. Antibody against EGFP was used and three replicates were analysed.

铁死亡（ferroptosis）是一种由脂质过氧化积累介导的受调控细胞死亡形式。越来越多的研究证据表明，铁死亡可在相邻细胞间发生扩散。然而，铁死亡扩散的潜在分子机制仍未明确。本研究证实，发生铁死亡的细胞会伸出细胞突起并释放大量细胞外囊泡（extracellular vesicles, EVs）。蛋白质组学分析显示，这些细胞外囊泡呈CD9阳性，并携带丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路的核心组分。细胞外囊泡转运的MAP2K1/2可增强靶细胞对铁死亡的敏感性。本研究进一步发现，与MAP2K相互作用的转录因子——过氧化物酶体增殖物激活受体γ（peroxisome-proliferator activator receptor gamma, PPARG）——可激活铁死亡防御系统的多种组分。此外，在活体中干扰基于细胞外囊泡的转运过程，可保护邻近组织免于发生铁死亡。本研究揭示了铁死亡细胞与周围邻近细胞之间存在依赖于细胞外囊泡的信号转运机制，并证实受MAPK调控的PPARG是受体细胞中铁死亡防御的关键分子。实验设计概况：本研究使用表达PPARG-增强绿色荧光蛋白（enhanced green fluorescent protein, EGFP）融合蛋白的U-2 OS细胞开展CUT&Tag测序，以分析PPARG的靶调控基因；实验采用抗EGFP抗体，共设置3次生物学重复并完成测序分析。