mTOR in programmed cell death and its therapeutic implications

Mechanistic target of rapamycin (mTOR), a highly conserved serine/threonine kinase, is involved in cellular metabolism, protein synthesis, and cell death. Programmed cell death (PCD) assists in eliminating aging, damaged, or neoplastic cells, and is indispensable for sustaining normal growth, fighting pathogenic microorganisms, and maintaining body homeostasis. mTOR has crucial functions in the intricate signaling pathway network of multiple forms of PCD. mTOR can inhibit autophagy, which is part of PCD regulation. Cell survival is affected by mTOR through autophagy to control reactive oxygen species production and the degradation of pertinent proteins. Additionally, mTOR can regulate PCD in an autophagy-independent manner by affecting the expression levels of related genes and phosphorylating proteins. Therefore, mTOR acts through both autophagy-dependent and -independent pathways to regulate PCD. It is conceivable that mTOR exerts bidirectional regulation of PCD, such as ferroptosis, according to the complexity of signaling pathway networks, but the underlying mechanisms have not been fully explained. This review summarizes the recent advances in understanding mTOR-mediated regulatory mechanisms in PCD. Rigorous investigations into PCD-related signaling pathways have provided prospective therapeutic targets that may be clinically beneficial for treating various diseases.     

LncRNA Kcnq1ot1/miR-214-3p/caspase-1通路调控高糖处理的心脏成纤维细胞焦亡

摘要 目的：探讨长链非编码RNA Kcnq1ot1在高糖处理的心脏成纤维细胞中调控焦亡的作用及具体机制。方法：培养C57BL/6乳鼠原代心脏成纤维细胞,分别用5.5 mM和30 mM葡萄糖培养,用免疫荧光、qRT-PCR和western blot方法检测NLRP3、caspase-1和IL-1β的表达。高糖处理的成纤维细胞抑制Kcnq1ot1,检测caspase-1的表达。生物信息学和荧光素酶报告基因检测验证与Kcnq1ot1和caspase-1存在共同互补结合位点的microRNA。应用qRT-PCR和western blot方法检测高糖诱导的心脏成纤维细胞干扰Kcnq1ot1后miR-214-3p的表达,以及过表达或干扰miR-214-3p后caspase-1的表达水平。高糖诱导的细胞单独干扰Kcnq1ot1或同时抑制Kcnq1ot1和miR-214-3p,检测caspase-1、NLRP3和IL-1?茁的表达水平。结果：高糖诱导的成纤维细胞中焦亡激活,Kcnq1ot1表达明显升高;抑制Kcnq1ot1后caspase-1表达显著下调。生物信息学和荧光素酶报告基因检测发现miR-214-3p与Kcnq1ot1和caspase-1存在共同互补结合位点。高糖诱导的心脏成纤维细胞干扰Kcnq1ot1后miR-214-3p表达升高;过表达 miR-214-3p 后caspase-1表达降低,抑制miR-214-3p后caspase-1表达升高。同时抑制Kcnq1ot1和miR-214-3p可逆转干扰Kcnq1ot1对caspase-1的降低作用。结论：干扰Kcnq1ot1能够通过抑制miR-214-3p/caspase-1信号转导通路,抑制高糖诱导的心脏成纤维细胞焦亡。     

Pyroptosis in Kidney Disease

In the last several decades, apoptosis interference has been considered clinically irrelevant in the context of renal injury. Recent discovery of programmed necrotic cell death, including necroptosis, ferroptosis, and pyroptosis refreshed our understanding of the role of cell death in kidney disease. Pyroptosis is characterized by a lytic pro- inflammatory type of cell death resulting from gasdermin-induced membrane permeabilization via activation of inflammatory caspases and inflammasomes. The danger-associated molecular patterns (DAMPs), alarmins and pro-inflammatory cytokines are released from pyroptotic cells in an uncontrolled manner, which provoke inflammation, resulting in secondary organ or tissue injuries. The caspases and inflammasome activation-related proteins and pore-forming effector proteins known as GSDMD and GSDME have been implicated in a variety of acute and chronic microbial and non-microbial kidney diseases. Here, we review the recent advances in pathological mechanisms of pyroptosis in kidney disease and highlight the potential therapeutic strategies in future.     

NLRP3-Caspase-1通路在小胶质细胞与H37Ra共培养模型中的作用及钾离子对其的影响

目的:在小胶质细胞与H37Ra结核菌株共培养模型中,探讨NLRP3-Caspase-1通路是否参与到小胶质细胞的损伤过程中,并观察钾离子对该过程的影响。方法:将H37Ra菌株与大鼠小胶质细胞共培养以模拟结核杆菌中枢神经系统感染造成的损伤,并通过real-time PCR,Western blot,ELISA,MTT等相关方法评估该过程中Caspase-1,NLRP3,IL-1β,IL-18等的基因转录及蛋白表达、分泌的变化规律;随后给予细胞外高钾干预,观察其对该模型中NLRP3-Caspase-1相关通路的影响。结果:通过小胶质细胞与H37Ra共培养:(1)小胶质细胞中NLRP3,Caspase-1,IL-1β及IL-18等的转录,表达及活化较前明显增加;(2)在应用细胞外高钾干预后,小胶质细胞中活性Caspase-1明显减少,同时其下游的活性IL-1β、IL-18产生及分泌也明显减少。结论:小胶质细胞与H37Ra共培养后,NLRP3-Caspase-1信号通路被激活,而通过细胞外高钾的干预能够抑制该过程中Caspase-1的活化,以及下游的IL-1β、IL-18的产生及分泌。     

Mechanisms of Gasdermin Family Members in Inflammasome Signaling and Cell Death

The Gasdermin (GSDM) family consists of Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C (GSDMC), Gasdermin D (GSDMD), Gasdermin E (GSDME) and Pejvakin (PJVK). GSDMD is activated by inflammasome-associated inflammatory caspases. Cleavage of GSDMD by human or mouse caspase-1, human caspase-4, human caspase-5, and mouse caspase-11 liberates the N-terminal effector domain from the C-terminal inhibitory domain. The N-terminal domain oligomerizes in the cell membrane and forms a pore of 10–16?nm in diameter, through which substrates of a smaller diameter, such as interleukin-1β and interleukin-18, are secreted. The increasing abundance of membrane pores ultimately leads to membrane rupture and pyroptosis, releasing the entire cellular content. Other than GSDMD, the N-terminal domain of all GSDMs, with the exception of PJVK, have the ability to form pores. There is evidence to suggest that GSDMB and GSDME are cleaved by apoptotic caspases. Here, we review the mechanistic functions of GSDM proteins with respect to their expression and signaling profile in the cell, with more focused discussions on inflammasome activation and cell death.     

人类免疫缺陷病毒感染者免疫功能重建不全的研究进展

约20%的人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染者在接受抗病毒治疗后,外周血CD4^＋ T细胞水平无法有效恢复,即免疫功能重建不全,但发生机制目前尚不明确。本文根据HIV感染者的临床和免疫学指标,分析发生免疫功能重建不全的风险因素;进一步从外周循环系统中T细胞损伤、细胞因子变化及肠道黏膜局部CD4^＋ T细胞删除等方面解析可能机制,其中肠道黏膜CD4^＋ T细胞删除可能与肠道微生物菌群诱导的细胞焦亡有关。虽然目前国际上有多项关于免疫功能重建不全患者治疗方案的临床试验,但由于缺乏一致的、有力的基础研究证据,导致疾病诊断指标缺乏,单独细胞因子给药治疗亦未取得突破性进展。因此,应深入探索免疫功能重建不全发生机制,进行大样本、长时间的前瞻性队列研究,制定免疫功能重建不全的临床与免疫学界定标准,从而为临床治疗提供科学依据。     

Mitochondrial Protein UCP1 Inhibits the Malignant Behaviors of Triple-negative Breast Cancer through Activation of Mitophagy and Pyroptosis

Triple-negative breast cancer (TNBC) is a massive threat to women''s health due to its high morbidity, malignancy, and the refractory, effective therapeutic option of TNBC is still deficient. The mitochondrial protein showed therapeutic potential on breast cancer, whereas the mechanism and downstream pathway of mitochondrial uncoupling protein 1 (UCP1) was not fully elucidated. We found that UCP1 was negatively regulated to the process of TNBC. Overexpressing UCP1 could inhibit the proliferation and metastasis of TNBC, meanwhile inducing the mitochondrial swelling and activation of mitophagy in vitro. Mitophagy activation was then assessed to elucidate whether it was downstream of UCP1 in TNBC metastasis. GSDME is the core of pyroptosis. We found that GSDME was activated in the TNBC cells when UCP1 levels were high. It regulates TNBC cell proliferation potential instead of the apoptosis process in vitro and in vivo. Our results suggested that UCP1 could inhibit the process of TNBC by activating mitophagy and pyroptosis. Impaired activation of mitophagy weakens the regulation effect of UCP1 on metastasis of TNBC, similar to the impairment of GSDME activation on the proliferation regulation of UCP1 on TNBC. UCP1 might be a novel therapeutic target of TNBC.     

Major cell death pathways at a glance

Cell death is a crucial process during development, homeostasis and immune regulation of multicellular organisms, and its dysregulation is associated with numerous pathologies. Cell death is often induced upon pathogen infection as part of the defense mechanism, and pathogens have evolved strategies to modulate host cell death. In this review, we will discuss the molecular mechanisms and physiological relevance of four major types of programmed cell death, namely apoptosis, necrosis, autophagic cell death and pyroptosis.     

OmpA of Klebsiella pneumoniae ATCC 13883 induces pyroptosis in HEp-2 cells,leading to cell-cycle arrest and apoptosis

Klebsiella pneumoniae is an opportunistic pathogenic bacterium that commonly causes pneumonia in elderly people. OmpA, a toxin that is highly expressed in the outer membrane of the bacterium, is one of the primary factors implicated in the pulmonary pathogenesis of K. pneumoniae. To evaluate the associated pyroptosis mechanism of infection, the ompA gene was cloned, and the protein was expressed, extracted, and used to treat human larynx epithelial cells. We observed that OmpA induces reactive oxygen species production and cell-cycle arrest in the G2/M phase in host cells, leading to subsequent apoptosis. Moreover, OmpA was found to induce IL-1β and IL-18 production in host cells, resulting in caspase-1 activation, which simultaneously stimulated pyroptosis, thus leading to the death of the host cells. We next sought to examine differential gene expression via RNA sequencing to better elucidate the mechanisms associated with these cellular changes, and found that genes associated with these pathways were more highly expressed in OmpA-treated cells than in K. pneumoniae-infected cells. Thus, cell-cycle arrest, apoptosis, and pyroptosis may serve as the primary defenses employed by host cells against OmpA. These results provide novel insights into the host defense against K. pneumoniae infection.     

Schistosoma antigens as activators of inflammasome pathway: from an unexpected stimulus to an intriguing role

Parasites of the genus Schistosoma are organisms capable of living for decades within the definitive host. They interfere with the immune response by interacting with host’s receptors. In this review, we discuss from the first reports to the most recent discoveries regarding the ability of Schistosoma antigens in triggering intracellular receptors and inducing inflammasome activation.