Abstract：
Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations: BAF: bafilomycin A1; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; siCtrl: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a.
摘要：
间充质干细胞（MSCs）用于细胞治疗；尽管如此，它们的应用受到移植后在促炎微环境中的低存活率的限制。骨髓间充质干细胞的巨自噬/自噬激活构成了应激适应通路，促进细胞稳态。我们的蛋白质组学数据表明RUBCNL/PACER（RUN和富含半胱氨酸的结构域含有beclin1相互作用蛋白样），自噬的正调节因子,也参与了细胞死亡。因此，我们筛选了在RUBCNL功能丧失或获得的情况下各种细胞死亡刺激下MSC的存活。当RUBCNL表达时,保护MSC免受TNF(肿瘤坏死因子)诱导的细胞死亡的影响。TNF通过诱导RIPK1激酶依赖性细胞凋亡或坏死促进炎症。我们确定，当半胱天冬酶失活时，MSC在TNF感应和坏死后屈服于RIPK1激酶依赖性凋亡。我们表明RUBCNL是RIPK1依赖性凋亡和坏死的负调节因子。此外，RUBCNL突变体失去调节自噬的能力，保留它们在负调节细胞死亡中的功能。我们还发现RUBCNL与RIPK1形成复合物，RIPK1响应TNF而分解。根据这一发现，RUBCNL表达限制RIPK1-TNFRSF1A/TNFR1复合物I的组装，表明RUBCNL和RIPK1之间的复合物形成抑制TNF信号传导。这些结果为RIPK1介导的细胞死亡和自噬机制之间的串扰提供了新的见解，并表明RUBCNL,由于其在自噬和凋亡/坏死中的功能二重性，可以靶向提高MSCs的治疗效果。
