稳定表达RFP-GFP-LC3的大鼠胰岛β细胞株的构建

为了简便自噬相关机理药物在胰岛β细胞上的高通量筛选,实验通过慢病毒转染技术,将RFP-GFP-LC3质粒转入大鼠胰岛β细胞株(RIN-m5f),用G418对感染细胞进行筛选,激光共聚焦进行活细胞拍摄验证,得到100%表达RFP-GFP-LC3质粒的细胞株。无血清饥饿处理细胞,结果显示:饥饿组GFP/RFP荧光点比值较对照组下降, P62蛋白表达量以及S6K磷酸化水平降低,加入10μmol/L氯喹部分抑制自噬后, GFP/RFP荧光点比值回升。以上结果表明稳定表达RFP-GFP-LC3的RIN-m5f构建成功,并能够以简单的检测方式准确表达自噬全过程,为自噬在糖尿病药物机理方面的研究奠定了基础。     

低频超声联合微泡造影剂通过激活自噬和凋亡对乳腺癌细胞活力的影响

自噬和凋亡是乳腺癌细胞数量和活力减少的重要因素,低频超声对肿瘤细胞的作用引起了研究者们的广泛兴趣,但是低频超声对乳腺癌细胞自噬和凋亡的作用尚不清楚。该文采用功率0.5 W/cm2的1 MHz低频超声联合微泡造影剂,作用于人乳腺癌细胞株MDA-MB-231 60s后,吖啶橙染色,电镜观察自噬的数量,Western blot检测微管相关蛋白轻链3-II(microtubule associated protein1 light chain 3-II,LC3-II)、自噬相关基因5(autophagy related 5,ATG5)和SQSTM1(sequestosome 1)/p62蛋白质水平变化,分别分析自噬的水平。Western blot检测caspase-3,膜联蛋白V/PI染色和DAPI染色方法分析MDA-MB-231细胞凋亡水平。ATG5 si RNA转染细胞可抑制自噬,caspase抑制剂Z-VAD-FMK可抑制凋亡,使用CCK-8分析自噬和凋亡对MDA-MB-231细胞的作用。结果显示,低频超声联合微泡造影剂促使LC3-II和ATG5蛋白质表达水平显著升高,而使SQSTM1/p62蛋白质表达水平显著下降(P0.05)。透射电镜和共聚焦观察发现,MDA-MB-231细胞自噬体数量增加。低频超声联合微泡造影剂促使caspase-3蛋白质表达水平升高,凋亡率增加。抑制自噬和凋亡后均明显缓解低频超声联合微泡造影剂对细胞活力的抑制作用(P0.05)。该研究结果说明,低频超声联合微泡造影剂通过激活自噬和凋亡抑制乳腺癌细胞株MDA-MB-231的增殖活力。     

饥饿及雨蛙素诱导的AR42J 细胞中自噬基因LC3及beclin-1 表达的变化

目的:观察饥饿及雨蛙素诱导的大鼠胰腺腺泡细胞AR42J中自噬基因LC3及beclin-1表达的变化,初步探讨吞噬(autophagy)在急性胰腺炎中的作用。方法:选择体外培养的生长状态良好的大鼠胰腺腺泡AR42J细胞,随机分为3组,饥饿组(N=10),雨蛙素处理组(N=10),空白对照组(N=10)。饥饿组加入充足的平衡盐溶液,雨蛙素处理组加入含10-7mol/L雨蛙素的全营养培养液,空白对照组加入含20%灭活胎牛血清的F12-K培养液(p H7.2-7.4),各组分别于处理后2、4、6 h收集细胞并提取蛋白质。采用免疫印迹法检测三组不同时点胰腺腺泡细胞AR42J中自噬基因Beclin-1和LC3的蛋白表达。结果:空白对照组不同时点beclin-1和LC3-II均呈低表达,且各时点比较差异无统计学意义(P<0.05)。饥饿组和雨蛙素处理组beclin-1和LC3-II的表达随处理时间的延长逐渐增加,且不同时点beclin-1和LC3-II的表达均较空白对照组显著增高,差异均具统计学意义(P<0.05)。结论:雨蛙素和饥饿刺激可导致大鼠胰腺腺泡细胞AR42J中LC3-II及beclin-1蛋白表达随作用时间的延长而增加,自噬可能参与了胰腺炎的发生发展过程。     

紫檀芪通过激活自噬缓解脊髓神经元细胞氧化应激损伤的作用及其机制

自噬在保护脊髓神经元细胞氧化应激损伤中具有重要的作用。紫檀芪(pterostilbene,PTE)是具有抗氧化作用的天然植物的提取物,但其对神经元细胞的作用及其机制尚不清楚。该文采用CCK-8分析PTE对大鼠原代脊髓神经元细胞的细胞毒性;不同浓度PTE作用神经元细胞24 h和48 h,透射电镜和Western blot检测微管相关蛋白轻链3-II(microtubule-associated protein 1 light chain3-II,MAP1LC3-II)、Beclin-1和P62蛋白质水平并分析自噬水平。PTE处理H2O2作用下的神经元细胞24 h,Western blot检测LC3-II水平,GFP-LC3转染观察自噬的数量。2′,7′-二氯二氢荧光素乙酰乙酸(2′,7′-dichloro-djhydrofl uorescein diacetate,DCFDA)和Mito SOX染色分析细胞活性氧(reactive oxygen species,ROS)水平,自噬相关基因5(autophagy related gene 5,ATG5)si RNA转染分析自噬在其中的作用。结果显示,20μmmol/L PTE对于神经元细胞无细胞毒性,PTE作用下神经元细胞中LC3-II、Beclin-1蛋白质水平呈剂量依懒性升高,而P62则呈剂量依懒性下降(P0.05)。PTE增加H2O2作用下的神经元细胞中LC3-II蛋白质水平(P0.05),自噬体数量增加,PTE可提高神经元细胞中自噬体数量。PTE明显降低神经元细胞中ROS水平,但ATG5 si RNA转染抑制自噬后显著逆转PTE的保护作用。该研究结果提示,PTE可能通过提高氧化应激状态下的脊髓神经元细胞自噬水平来抑制细胞ROS的产生。     

高糖环境下beclin2介导的自噬增强绒毛膜滋养层细胞凋亡

目的探讨高糖对人绒毛膜滋养层细胞早期凋亡及自噬水平的影响。方法取足月妊娠的正常及妊娠期糖尿病(gestational diabetes mellitus,GDM)患者的胎盘组织,透射电镜观察滋养层细胞超微结构的改变。体外用不同浓度的含糖培养基培养滋养层细胞系HTR8/Svneo细胞24h,分为低糖组(1.57mmol/L或LG2 2.25mmol/L),正常对照组(5.57mmol/L)和高糖组(10.57mmol/L、15.57mmol/L或40.57mmol/L);流式细胞术检测细胞早期凋亡率;q-PCR检测细胞内beclin1、beclin2和ATG7等自噬相关基因的m RNA表达水平;Western blot检测Ⅱ型自噬标志蛋白微管相关蛋白1轻链3(LC3-II)和p62蛋白表达水平。结果透射电镜下可见GDM组滋养层细胞中存在自噬小体且空泡数目明显多于正常足月妊娠组,其游离面微绒毛出现明显倒覆及坍塌现象;在体外培养的滋养层细胞中,流式细胞术检测显示1.57mmol/L低糖组及40.57mmol/L高糖组较正常对照组HTR8/SVneo细胞的早期凋亡率均明显增加;q-PCR分析发现40.57mmol/L高糖组beclin2及ATG7 m RNA表达水平较正常对照组升高,beclin1 m RNA表达无差异;Western blot检测表明,与正常对照组比较,40.57mmol/L高糖组LC3-II蛋白表达水平升高,p62蛋白表达水平降低。结论高糖可通过beclin2介导的自噬信号途径增强滋养层细胞自噬水平进而导致细胞凋亡,并可能与不良的妊娠结局相关。     

几个能量代谢相关蛋白在HeLa细胞自噬过程中表达下调

自噬是真核细胞中的一种保守的代谢信号通路。人们已经知道自噬与肿瘤发生等疾病密切相关,但对于自噬的分子机制仍然不是很清楚。鉴定更多的自噬相关蛋白对于进一步阐明自噬的分子机制具有重要意义。该研究使用饥饿法处理HeLa细胞,通过电镜观察以及检测自噬标记蛋白LC3-I的转换,证实HeLa细胞发生了明显的自噬。之后,使用双向电泳结合串联质谱分析鉴定细胞自噬时发生变化的蛋白质。结果发现果糖二磷酸醛缩酶A、GAPDH和ATP合成酶O亚基的量在HeLa细胞发生自噬后明显降低。实时定量PCR结果证明饥饿诱导后,这三种蛋白的mRNA水平都发生了明显的下降。使用自噬抑制剂3-Methyladenine预处理HeLa细胞后再行饥饿,三种蛋白mRNA的表达水平与正常细胞相当而明显高于饥饿诱导的细胞。结果表明这三种蛋白在饥饿诱导的自噬中表达下调,其分子机制还有待进一步研究。     

自噬在熊果酸诱导前列腺癌PC3细胞凋亡中的作用机制研究

为了探讨自噬在熊果酸抑制前列腺癌PC3细胞凋亡中的作用机制,PC3细胞培养至对数生长期后,以无糖、无氨基酸培养液代替原培养液培养细胞,并用不同浓度的熊果酸进行干预。72 h后收集细胞,采用透射电镜和免疫荧光技术观察PC3细胞自噬情况;Western Blot检测ATG5和Beclin-1蛋白表达;Elisa法测定Caspase-3、Caspase-8和Caspase-9含量;流式细胞技术检测PC3细胞凋亡情况。结果表明,PC3细胞饥饿72 h后,细胞自噬明显增强。与对照组比较,熊果酸作用后的细胞自噬程度明显减弱;ATG5和Beclin-1蛋白表达显著减少(P0.05);Caspase-3、Caspase-8和Caspase-9含量显著增加(P0.05);PC3细胞凋亡率极显著升高(P0.01)。实验初步揭示,熊果酸可通过抑制饥饿状态的前列腺癌PC3细胞自噬,并进一步通过促进凋亡因子的分泌诱导其凋亡。     

真菌次级代谢产物11’-deoxyverticillin A(C42)通过降低自噬基因的表达抑制肝炎病毒(HBV)复制

C42属于epipolythiodioxopiperazines(ETPs)二酮呱嗪类化合物,具有多种生物学活性,包括抑制病毒复制;不过其对hepatitis B virus(HBV)复制的影响及机理鲜有报道。自噬是广泛存在于真核细胞、通过溶酶体降解长半衰期蛋白的现象,参与多种生理、病理过程。有研究发现自噬对HBV的复制至关重要。C42是否通过改变自噬来影响此病毒的复制目前还未见报道。在该研究中,我们发现表达HBV基因组的Hep G2.215细胞较原始的Hep G2细胞,自噬体明显增加并伴随着Akt磷酸化的增高。C42可以降低自噬基因LC3-II和p62的水平,同时会影响Akt信号通路。氯喹是一种自噬抑制剂,它的存在可以抑制C42导致的LC3-II降低,表明C42可以引起该细胞的自噬。敲降自噬基因和抑制Akt磷酸化均可以减少HBV-X蛋白表达。而利用氯喹抑制自噬体与溶酶体的融合却提高了HBV-X蛋白水平。由于HBV-X对该病毒的复制至关重要,因此,我们认为,C42通过自噬和Akt信号通路来抑制HBV的复制。     

真菌次级代谢产物11’-deoxyverticillin A（C42）通过降低自噬基因的表达抑制肝炎病毒（HBV）复制

C42属于epipolythiodioxopiperazines（ETPs）二酮呱嗪类化合物,具有多种生物学活性,包括抑制病毒复制;不过其对hepatitis B virus（HBV）复制的影响及机理鲜有报道。自噬是广泛存在于真核细胞、通过溶酶体降解长半衰期蛋白的现象,参与多种生理、病理过程。有研究发现自噬对HBV的复制至关重要。C42是否通过改变自噬来影响此病毒的复制目前还未见报道。在该研究中,我们发现表达HBV基因组的HepG2.215细胞较原始的HepG2细胞,自噬体明显增加并伴随着Akt磷酸化的增高。C42可以降低自噬基因LC3-II和p62的水平,同时会影响Akt信号通路。氯喹是一种自噬抑制剂,它的存在可以抑制C42导致的LC3-II降低,表明C42可以引起该细胞的自噬。敲降自噬基因和抑制Akt磷酸化均可以减少HBV-X蛋白表达。而利用氯喹抑制自噬体与溶酶体的融合却提高了HBV-X蛋白水平。由于HBV-X对该病毒的复制至关重要,因此,我们认为,C42通过自噬和Akt信号通路来抑制HBV的复制。     

RTN4对于结肠癌细胞增殖的调控作用

2018年全球癌症统计调查显示,结直肠癌约占患癌新病例的12.1%。因此,寻找新的结肠癌发生有关的基因,发现新的治疗靶点显得尤为迫切。通过数据库分析发现,RTN4基因的表达水平与结肠癌患者生存率的相关性具有统计学意义。针对RTN4基因构建其干扰质粒,将慢病毒作为载体转染结肠癌HCT116细胞中构建敲低RTN4的结肠癌细胞系,最后检测了低表达后RTN4基因的细胞增殖。结果发现,敲低RTN4基因后显著促进了结肠癌细胞HCT116的增殖,研究通过Western blot观察敲低RTN4后HCT116细胞自噬通路相关蛋白p62和LC3的表达情况,发现与对照组相比较,敲低RTN4组LC3转化量（LC3-II/LC3-I）增多,而p62蛋白减少。研究分析了RTN4的潜在抑癌作用,发现敲低RTN4基因会显著增强结肠癌细胞的增殖能力,并且诱导自噬,说明RTN4可能与激活LC3/p62自噬途径有关。     

The Anticancer Agent Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Prosurvival Autophagy by Two Mechanisms: PERSISTENT INDUCTION OF AUTOPHAGOSOME SYNTHESIS AND IMPAIRMENT OF LYSOSOMAL INTEGRITY

Autophagy functions as a survival mechanism during cellular stress and contributes to resistance against anticancer agents. The selective antitumor and antimetastatic chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) causes lysosomal membrane permeabilization and cell death. Considering the integral role of lysosomes in autophagy and cell death, it was important to assess the effect of Dp44mT on autophagy to further understand its mechanism of action. Notably, Dp44mT affected autophagy by two mechanisms. First, concurrent with its antiproliferative activity, Dp44mT increased the expression of the classical autophagic marker LC3-II as a result of induced autophagosome synthesis. Second, this effect was supplemented by a reduction in autophagosome degradation as shown by the accumulation of the autophagic substrate and receptor p62. Conversely, the classical iron chelator desferrioxamine induced autophagosome accumulation only by inhibiting autophagosome degradation. The formation of redox-active iron or copper Dp44mT complexes was critical for its dual effect on autophagy. The cytoprotective antioxidant N-acetylcysteine inhibited Dp44mT-induced autophagosome synthesis and p62 accumulation. Importantly, Dp44mT inhibited autophagosome degradation via lysosomal disruption. This effect prevented the fusion of lysosomes with autophagosomes to form autolysosomes, which is crucial for the completion of the autophagic process. The antiproliferative activity of Dp44mT was suppressed by Beclin1 and ATG5 silencing, indicating the role of persistent autophagosome synthesis in Dp44mT-induced cell death. These studies demonstrate that Dp44mT can overcome the prosurvival activity of autophagy in cancer cells by utilizing this process to potentiate cell death.     

A tethering coherent protein in autophagosome maturation

Autophagy is a cellular pathway that degrades damaged organelles, cytosol and microorganisms, thereby maintaining human health by preventing various diseases including cancers, neurodegenerative disorders and diabetes. In autophagy, autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. The proper autophagosome-lysosome fusion is pivotal for efficient autophagy activity. However, the molecular mechanism that specifically directs the fusion process is not clear. Our study reported that lysosome-localized TECPR1 (TECtonin β-Propeller Repeat containing 1) binds the autophagosome-localized ATG12–ATG5 conjugate and recruits it to autolysosomes. TECPR1 also binds PtdIns3P in an ATG12–ATG5-dependent manner. Consequently, depletion of TECPR1 leads to a severe defect in autophagosome maturation. We propose that the interaction between TECPR1 and ATG12–ATG5 initiates the fusion between the autophagosome and lysosome, and TECPR1 is a TEthering Coherent PRotein in autophagosome maturation.     

Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.     

Autophagosome Requires Specific Early Sec Proteins for Its Formation and NSF/SNARE for Vacuolar Fusion

Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.     

RNA-binding protein,human antigen R regulates hypoxia-induced autophagy by targeting ATG7/ATG16L1 expressions and autophagosome formation

Autophagy, a prosurvival mechanism offers a protective role during acute kidney injury. We show novel findings on the functional role of RNA binding protein, HuR during hypoxia-induced autophagy in renal proximal tubular cells-2 (HK-2). HK-2 cells showed upregulated expressions of HuR and autophagy-related proteins such as autophagy related 7 (ATG7), autophagy related 16 like 1 (ATG16L1), and LC3II under hypoxia. Increased autophagosome formation was visualized as LC3 puncta in hypoxic cells. Further, short hairpin-RNA-mediated loss of HuR function in HK-2 cells significantly decreased ATG7 and ATG16L1 protein expressions. Bioinformatics prediction revealed HuR motif binding on the coding region of ATG7 and AU-rich element at 3′UTR ATG16L1 messnger RNA (mRNA). The RNA immunoprecipitation study showed that HuR was predominantly associated with ATG7 and ATG16L1 mRNAs under hypoxia. In addition, HuR enhanced autophagosome formation by regulating LC3II expressions. These results show that HuR regulates ATG7 and ATG16L1 expressions and thereby mediate autophagy in HK-2 cells. Importantly, HuR knockdown cells underwent apoptosis during hypoxia as observed through the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Collectively, these findings show the crucial role of HuR under hypoxia by regulating autophagy and suppressing apoptosis in renal tubular cells.     

A tethering coherent protein in autophagosome maturation

Autophagy is a cellular pathway that degrades damaged organelles, cytosol and microorganisms, thereby maintaining human health by preventing various diseases including cancers, neurodegenerative disorders and diabetes. In autophagy, autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. The proper autophagosome-lysosome fusion is pivotal for efficient autophagy activity. However, the molecular mechanism that specifically directs the fusion process is not clear. Our study reported that lysosome-localized TECPR1 (TECtonin β-Propeller Repeat containing 1) binds the autophagosome-localized ATG12-ATG5 conjugate and recruits it to autolysosomes. TECPR1 also binds PtdIns3P in an ATG12-ATG5-dependent manner. Consequently, depletion of TECPR1 leads to a severe defect in autophagosome maturation. We propose that the interaction between TECPR1 and ATG12-ATG5 initiates the fusion between the autophagosome and lysosome, and TECPR1 is a TEthering Coherent PRotein in autophagosome maturation.     

Monitoring autophagy in wheat living cells by visualization of fluorescence protein-tagged ATG8

Autophagy is a highly conserved eukaryotic degradation process during which bulk cytoplasmic materials are transported by double-membrane autophagosomes into the vacuole for degradation. Methods of monitoring autophagy are indispensable in studying the mechanism and functions of autophagy. AuTophaGy-related protein 8 (ATG8) functions in autophagosome assembly by decorating on autophagic membranes, and the inner membrane-bound ATG8 proteins enter the vacuole via active autophagy flux. Fluorescence protein (FP)-tagged forms of ATG8 have been explored as visual markers to monitor autophagy in animals and several plant species. Here, we evaluated and modified this FP-ATG8-based autophagy monitoring method in wheat (Triticum aestivum L.) by fluorescence observation of green fluorescence protein (GFP)-tagged and Discosoma red fluorescent protein (DsRED)-tagged forms of one wheat ATG8, TaATG8h, in wheat mesophyll protoplasts. Under a nutrient-starvation condition, punctate GFP/DsRED- TaATG8h fluorescence representing autophagosomes was clearly observed in the cytoplasm. The accumulation of GFP-TaATG8h-labeled autophagosomes was impaired by the autophagosome biogenesis inhibitor 3-methyladenine but enhanced by the vacuolar degradation inhibitor concanamycin A. In addition, accumulated spreading fluorescence was observed in the vacuole, indicating active autophagy fluxes which led to continuous degradation of GFP/DsRED-TaATG8h fusions and release of protease-tolerant free GFP/DsRED proteins in the vacuole. To observe FP-tagged TaATG8h in other types of wheat cell, we also expressed GFP-TaATG8h in leaf epidermal cells. Consistent with its performance in protoplasts, GFP-TaATG8h showed punctate fluorescence representing autophagosomes in leaf epidermal cells. Taken together, our results proved the feasibility of using FP-tagged ATG8 to monitor both autophagosome accumulation and autophagy flux in living wheat cells.     

CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation†

The macroautophagy/autophagy and inflammasome pathways are linked through their roles in innate immunity and chronic inflammatory disease. Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates pro-inflammatory eicosanoid production. Whether C1P also regulates autophagy and inflammasome assembly/activation is not known. Here we show that CPTP (a protein that traffics C1P from its site of phosphorylation in the trans-Golgi to target membranes) regulates both autophagy and inflammasome activation. In human epithelial cells, knockdown of CPTP (but not GLTP [glycolipid transfer protein]) or expression of C1P binding-site point mutants, stimulated an 8- to 10-fold increase in autophagosomes and altered endogenous LC3-II and SQSTM1/p62 protein expression levels. CPTP depletion-induced autophagy elevated early markers of autophagosome formation (Golgi-derived ATG9A-vesicles, WIPI1), required key phagophore assembly and elongation factors (ATG5, ATG7, ULK1), and suppressed MTOR phosphorylation and that of its downstream target, RPS6KB1/p70S6K. Wild-type CPTP overexpression exerted a protective effect against starvation-induced autophagy. In THP-1 macrophage-like surveillance cells, CPTP knockdown induced not only autophagy but also elevated CASP1/caspase-1 levels, and strongly increased IL1B/interleukin-1β and IL18 release via a NLRP3 (but not NLRC4) inflammasome-based mechanism, while only moderately increasing inflammatory (pyroptotic) cell death. Inflammasome assembly and activation stimulated by CPTP depletion were autophagy dependent. Elevation of intracellular C1P by exogenous C1P treatment (instead of CPTP inhibition) also induced autophagy and IL1B release. Our findings identify human CPTP as an endogenous regulator of early-stage autophagosome assembly and inflammasome-driven, pro-inflammatory cytokine generation and release.     

Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A₁, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response.