GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation

Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.     

Silencing FUNDC1 alleviates chronic obstructive pulmonary disease by inhibiting mitochondrial autophagy and bronchial epithelium cell apoptosis under hypoxic environment

Chronic obstructive pulmonary disease (COPD) is a major global epidemic with increasing incidence worldwide. The pathogenesis of COPD is involved with mitochondrial autophagy. Recently, it has been reported that FUN14 domain containing 1 (FUNDC1) is a mediator of mitochondrial autophagy. Therefore, we hypothesized that FUNDC1 was involved in cigarette smoke (CS)-induced COPD progression by regulating mitochondrial autophagy. In vitro cigarette smoke extract (CSE)-treated human bronchial epithelial cell (hBEC) Beas-2B cell line and in vivo CS-induced COPD mouse models were developed, in which FUNDC1 expression was measured. Next, whether FUNDC1 interacted with dynamin-related protein 1 (DRP1) in COPD was investigated. The functional mechanism of FUNDC1 in COPD was evaluated through gain- or loss-of-function studies. Then, pulmonary function, mitochondrial transmembrane potential (MTP) and mucociliary clearance (MCC) were examined. Levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and expression of autophagy-specific markers (light chain 3 [LC3] II, LC3 I, and Tom20) were measured. Finally, apoptosis and mitochondrial autophagy were assessed. FUNDC1 was highly expressed in CSE-treated hBECs and COPD mice. Meanwhile, FUNDC1 was proved to interact with DRP1 in CSE-treated cells. Moreover, in CSE-treated hBECs, silencing FUNDC1 was observed to reduce levels of IL-6 and TNF-α, and MTP but increase MCC, and inhibit CSE-induced mitochondrial autophagy and Beas-2B cell apoptosis, which was consistent with the trend in COPD mouse models. In addition, pulmonary function of COPD mouse models was increased in response to FUNDC1 silencing. Finally, silencing of DRP1 also inhibited mitochondrial autophagy and Beas-2B cell apoptosis. Collectively, FUNDC1 silencing could suppress the progression of COPD by inhibiting mitochondrial autophagy and hBEC apoptosis through interaction with DRP1, highlighting a potential therapeutic target for COPD treatment.     

Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells

Accumulating evidence has shown that dysfunctional mitochondria can be selectively removed by mitophagy. Dysregulation of mitophagy is implicated in the development of neurodegenerative disease and metabolic disorders. How individual mitochondria are recognized for removal and how this process is regulated remain poorly understood. Here we report that FUNDC1, an integral mitochondrial outer-membrane protein, is a receptor for hypoxia-induced mitophagy. FUNDC1 interacted with LC3 through its typical LC3-binding motif Y(18)xxL(21), and mutation of the LC3-interaction region impaired its interaction with LC3 and the subsequent induction of mitophagy. Knockdown of endogenous FUNDC1 significantly prevented hypoxia-induced mitophagy, which could be reversed by the expression of wild-type FUNDC1, but not LC3-interaction-deficient FUNDC1 mutants. Mechanistic studies further revealed that hypoxia induced dephosphorylation of FUNDC1 and enhanced its interaction with LC3 for selective mitophagy. Our findings thus offer insights into mitochondrial quality control in mammalian cells.     

MicroRNA-1 facilitates hypoxia-induced injury by targeting NOTCH3

Cell proliferation, apoptosis, and autophagy have been reported to be related to myocardial ischemia injury. MicroRNAs have attracted wide attention on regulating cell proliferation, apoptosis, and autophagy. miR-1 expression has been reported to be dysregulated in cardiac tissue or cells with hypoxia, while the exact roles as well as underlying mechanism remain poorly understood. In this study, we investigated the potential roles of miR-1 in cell proliferation, apoptosis, and autophagy in hypoxia-treated cardiac injury and explored the underlying mechanism using H9c2 cells. Results showed that hypoxic stimulation inhibited cell proliferation and the expression of miR-1 but promoted cell apoptosis in H9c2 cells. Moreover, overexpression of miR-1 promoted cell apoptosis and inhibited cell proliferation and autophagy in H9c2 cells treated with hypoxia, while its knockdown played an opposite effect. In addition, bioinformatics, luciferase reporter, and RNA immunoprecipitation analyses indicated that NOTCH3 was a direct target of miR-1 and its upregulation reversed the effects of miR-1 on cell proliferation, apoptosis, and autophagy in hypoxia-treated H9c2 cells. Taken together, our data suggested that miR-1 promoted hypoxia-induced injury by targeting NOTCH3, indicating novel therapeutic targets for treatment of myocardial ischemia injury.     

Retracted: Deletion of long noncoding RNA EFNA3 aggravates hypoxia-induced injury in PC-12 cells by upregulation of miR-101a

Long noncoding RNAs (lncRNAs) have been reported to be involved in several neurological pathogenesis conditions including cerebral ischemia. In the current study, the functions of lncRNA EFNA3 on hypoxia-injured rat adrenal pheochromocytoma (PC-12) cells and the underlying molecular mechanism were studied. The expression of lncRNA EFNA3 was silenced by short hairpin RNA transfection, after which the cells were subjected with hypoxia. Cell viability, migration, invasion, and apoptosis were, respectively, determined by trypan blue staining, Transwell assay, annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining, and Western blot analysis. The cross regulation between lncRNA EFNA3 and miR-101a, as well as between miR-101a and Rho associated coiled-coil containing protein kinase 2 (ROCK2) were detected by performing quantitative real-time polymerase chain reaction, RNA pull-down, RNA immunoprecipitation, luciferase activity assay, and Western blot analysis. Studies showed that lncRNA EFNA3 was highly expressed in response to hypoxia. Deletion of lncRNA EFNA3 significantly aggravated hypoxia-induced injury in PC-12 cells, as the impairment of cell viability, migration, and invasion, and the inducement of apoptosis. LncRNA EFNA3 worked as a sponging molecule for miR-101a and miR-101a suppression-protected PC-12 cells against hypoxia-induced injury even when lncRNA EFNA3 was silenced. ROCK2 was a target gene of miR-101a. ROCK2 overexpression exhibited neuroprotective activities. Besides, ROCK2 overexpression activated Wnt/β-catenin pathway whereas it deactivated JAK/STAT pathway upon hypoxia. Our study suggests that deletion of lncRNA EFNA3 aggravates hypoxia-induced injury in PC-12 cells by upregulating miR-101a, which further targets ROCK2.     

Epigallocatechin gallate reduces hypoxia-induced apoptosis in human hepatoma cells

Cell detachment from extracellular matrix is closely related to induction of apoptosis. Epigallocatechin gallate (EGCG) has been shown to have antioxidant effect and to protect hypoxia-induced damage. We investigated whether EGCG reduced hypoxia-induced apoptosis and cell detachment in HepG2 cells. EGCG prevented cell death by hypoxia (0.5% O2) in a dose-dependent manner (hypoxic cell viability, 54.67%). RT-PCR and caspase3 activity assay showed that the hypoxia-induced cell death was caused by apoptosis increasing mRNA level of BAX, CASP3, and caspase3 activity. EGCG reduced increase of these mRNA and caspase3 activity. Western blot analysis and immunocytochemistry showed that EGCG increased cell adhesion proteins including E-cadherin (CDH1), tumor-associated calcium signal transducer 1 (TACSTD1), and protein tyrosine kinase 2 (PTK2) decreased by hypoxia. Hypoxia-induced apoptosis in HepG2 cells, and EGCG contributed to the HepG2 cell survival by attenuating the apoptosis.     

Autophagy protein Ulk1 promotes mitochondrial apoptosis through reactive oxygen species

Regardless of rapid progression in the field of autophagy, it remains a challenging task to understand the cross talk with apoptosis. In this study, we overexpressed Ulk1 in HeLa cells and evaluated the apoptosis-inducing potential of the Ulk1 gene in the presence of cisplatin. The gain of function of Ulk1 gene showed a decline in cell viability and colony formation in HeLa cells. The Ulk1-overexpressing cells showed higher apoptotic attributes by an increase in the percentage of annexin V, escalated expression of Bax/Bcl2 ratio, and caspase-9, -3/7 activities. Further, reactive oxygen species (ROS) generation was found to be much higher in HeLa-Ulk1 than in the mock group. Scavenging the ROS by N-acetyl-L-cysteine increased cell viability and colony number as well as mitochondrial membrane potential (MMP). Our data showed that Ulk1 on entering into mitochondria inhibits the manganese dismutase activity and intensifies the mitochondrial superoxide level. The Ulk1-triggered autophagy (particularly mitophagy) resulted in a fall in ATP; thus the nonmitophagic mitochondria overwork the electron-transport cycle to replenish energy demand and are inadvertently involved in ROS overproduction that led to apoptosis. In this present investigation, our results decipher a previously unrecognized perspective of apoptosis induction by a key autophagy protein Ulk1 that may contribute to identification of its tumor-suppressor properties through dissecting the connection among cellular bioenergetics, ROS, and MMP.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Shankar Sadasivan and Anu Waghray have contributed equally to this work.     

ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy

Autophagy eliminates dysfunctional mitochondria in an intricate process known as mitophagy. ULK1 is critical for the induction of autophagy, but its substrate(s) and mechanism of action in mitophagy remain unclear. Here, we show that ULK1 is upregulated and translocates to fragmented mitochondria upon mitophagy induction by either hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1, phosphorylating it at serine 17, which enhances FUNDC1 binding to LC3. A ULK1‐binding‐deficient mutant of FUNDC1 prevents ULK1 translocation to mitochondria and inhibits mitophagy. Finally, kinase‐active ULK1 and a phospho‐mimicking mutant of FUNDC1 rescue mitophagy in ULK1‐null cells. Thus, we conclude that FUNDC1 regulates ULK1 recruitment to damaged mitochondria, where FUNDC1 phosphorylation by ULK1 is crucial for mitophagy.     

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.     

Mammalian Ste20-like protein kinase 3 plays a role in hypoxia-induced apoptosis of trophoblast cell line 3A-sub-E

Mammalian Ste20-like protein kinase 3 (Mst3) is a key player in inducing apoptosis in a variety of cell types and has recently been shown to participate in the signaling pathway of hypoxia-induced apoptosis of human trophoblast cell line 3A-sub-E (3A). It is believed that oxidative stress may occur during hypoxia and induce the expression of Mst3 in 3A cells via the activation of c-Jun N-terminal protein kinase 1 (JNK1). This hypothesis was demonstrated by the suppressive effect of dl-α-lipoic acid, a reactive oxygen species scavenger, in hypoxia-induced responses of 3A cells such as Mst3 expression, nitrotyrosine formation, JNK1 activation and apoptosis. Similar results were also observed in trophoblasts of human placental explants in both immunohistochemical studies and immunoblot analyses. These suggested that the activation of Mst3 might trigger the apoptotic process in trophoblasts by activating caspase 3 and possibly other apoptotic pathways. The role of nitric oxide synthase (NOS) and NADPH oxidase (NOX) in hypoxia-induced Mst3 up-regulation was also demonstrated by the inhibitory effect of N(G)-nitro-l-arginine and apocynin, which inhibits NOS and NOX, respectively. Oxidative stress was postulated to be induced by NOS and NOX in 3A cells during hypoxia. In conclusion, hypoxia induces oxidative stress in human trophoblasts by activating NOS and NOX. Subsequently, Mst3 is up-regulated and plays an important role in hypoxia-induced apoptosis of human trophoblasts.     

Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosisresistant. Autophagy (cellular "self digestion") has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (<1% O(2)). Under normoxic conditions, all five cell lines undergo etoposide-induced apoptosis whereas hypoxia fails to induce these apoptotic responses. All five cell lines induce an autophagic response and undergo cell death in hypoxia. Hypoxia-induced cell death was reduced upon treatment with the autophagy inhibitor 3-methyladenine, but not with the caspase inhibitor z-VAD-fmk. By knocking down the autophagy proteins Beclin-1 or ATG5, hypoxia-induced cell death was also reduced. The pro-cell death Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19kDainteracting protein 3) is upregulated during hypoxia and is known to induce autophagy and cell death. We found that BNIP3 overexpression induced autophagy, while expression of BNIP3 siRNA or a dominant-negative form of BNIP3 reduced hypoxia-induced autophagy. Taken together, these results suggest that prolonged hypoxia induces autophagic cell death in apoptosis-competent cells, through a mechanism involving BNIP3.     

Ablation of FUNDC1-dependent mitophagy renders myocardium resistant to paraquat-induced ferroptosis and contractile dysfunction

Paraquat is a quaternary nitrogen herbicide evoking mitochondrial damage and heart failure with little therapeutic remedies available. Recent reports depicted a role for unchecked autophagy in paraquat-induced cardiotoxicity. This study was designed to examine the role of the mitophagy receptor protein FUNDC1 in paraquat-induced cardiac contractile and mitochondrial injury using a murine model of FUNDC1 knockout (FUNDC1^?/?) mice. WT and FUNDC1^?/? mice were challenged with paraquat (45 mg/kg, single injection, i.p.) for 72 h prior to examination of cardiac contractile and intracellular Ca²⁺ properties, mitochondrial integrity, mitochondrial function, O₂^? production, apoptosis, autosis and ferroptosis. Our results found that paraquat challenge compromised echocardiographic, contractile and intracellular Ca²⁺ properties in conjunction with mitochondrial damage (reduced levels of PGC1α, UCP2, NAD+, and citrate synthase activity along with fragmentation manifested by elevated Drp1 and TEM ultrastructural changes), the effects of which were overtly attenuated or obliterated by FUNDC1 ablation. Paraquat triggered ferroptosis, apoptosis (but not autosis) and unchecked mitophagy as evidenced by downregulation of GPx4, SLC7A11, Bcl2, TOM20 and ferritin as well as upregulated levels of Bax, TNFα, IL6, NCOA4 and FUNDC1, the effects of which were relieved by FUNDC1 ablation. Further study noted dephosphorylation of JNK upon paraquat challenge, the effect of which was obliterated by FUNDC1 knockout. In vitro evaluation of BODIPY ferroptosis and cardiomyocyte function revealed FUNDC1 ablation inhibited paraquat-induced increase in BODIPY lipid peroxidation and cardiomyocyte contractile dysfunction, the effects of which were nullified and mimicked by inhibition of JNK or ferroptosis and activation of JNK, respectively. Taken together, our data suggest an essential role for FUNDC1/JNK-mediated ferroptosis in paraquat exposure-evoked cardiac and mitochondrial injury.     

丹参酮ⅡA调节microRNA-1保护缺氧心肌细胞的作用研究

目的:研究丹参酮Ⅱ A(TanshinoneⅡA)通过调节microRNA-1抗心肌细胞缺氧损伤的作用。方法:原代培养新生大鼠心肌细胞,建立心肌细胞缺氧模型。MTT法检测心肌细胞存活率(%);TUNEL、流式细胞术测心肌细胞凋亡率;激光共聚焦检测心肌细胞内钙离子[Ca2+]i浓度的变化情况。结果:MTT结果显示丹参酮ⅡA对缺氧心肌细胞及过表达miR-1引起心肌细胞损伤具有保护作用。丹参酮ⅡA增加了缺氧心肌细胞的存活率(P0.05),同时给予丹参酮ⅡA和miR-1组与单独miR-1损伤组相比较,存活率也明显升高,呈现剂量依赖性。TUNEL结果显示丹参酮ⅡA可以抑制缺氧诱导的细胞凋亡,丹参酮ⅡA可以明显降低由缺氧导致的细胞凋亡率(P0.05)。共聚焦检测结果显示,缺氧损伤的心肌细胞内[Ca2+]i显著升高1322.72±5.16(vs正常对照组,P0.05),丹参酮ⅡA则有效抑制由缺氧引起过高的[Ca2+]i。miR-1诱导的细胞内[Ca2+]i升高至1349.33±62.63,约为正常对照组的1.96倍,而丹参酮ⅡA则有效抑制胞内过高的[Ca2+]i,从而发挥心肌保护作用。结论:丹参酮ⅡA可能是通过抑制胞内miR-1的表达,参与对钙离子浓度的调控,发挥其对心肌细胞的保护作用。     

Acetylcholine inhibits long-term hypoxia-induced apoptosis by suppressing the oxidative stress-mediated MAPKs activation as well as regulation of Bcl-2, c-IAPs,and caspase-3 in mouse embryonic stem cells

This study examined the effect of acetylcholine (ACh) on the hypoxia-induced apoptosis of mouse embryonic stem (ES) cells. Hypoxia (60 h) decreased both the cell viability and level of [³H] thymidine incorporation, which were prevented by a pretreatment with ACh. However, the atropine (ACh receptor [AChR] inhibitor) treatment blocked the protective effect of ACh. Hypoxia (90 min) increased the intracellular level of reactive oxygen species (ROS). On the other hand, ACh inhibited the hypoxia-induced increase in ROS, which was blocked by an atropine treatment. Subsequently, the hypoxia-induced ROS increased the level of p38 mitogen activated protein kinase (MAPK) and Jun-N-terminal kinase (JNK) phosphorylation, which were inhibited by the ACh pretreatment. Moreover, hypoxic exposure (90 min) increased the level of nuclear factor-κB (NF-κB) phosphorylation, which was blocked by a pretreatment with SB 203580 (p38 MAPK inhibitor) or SP 600125 (JNK inhibitor). However, hypoxia (60 h) decreased the protein levels of Bcl-2 and c-IAPs (cellular inhibitor of apoptosis proteins) but increased the level of caspase-3 activation. All these effects were inhibited by a pretreatment with ACh. In conclusion, ACh prevented the hypoxia-induced apoptosis of mouse ES cells by inhibiting the ROS-mediated p38 MAPK and JNK activation as well as the regulation of Bcl-2, c-IAPs, and caspase-3. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hypoxia-induced downregulation of autophagy mediator Beclin-1 reduces the susceptibility of malignant intestinal epithelial cells to hypoxia-dependent apoptosis

Disruption of tumor blood supply causes tumor hypoxia. Hypoxia can induce cell death, but cancer cells that remain viable in the absence of oxygen often possess an increased survival potential, and tumors formed by these cells tend to grow particularly aggressively. Thus, developing approaches aimed at increasing the susceptibility of malignant cells to hypoxia-induced death represents a potentially important avenue for cancer treatment. Molecular mechanisms that control the survival of cancer cells under hypoxia are not well understood. In an effort to understand them we found that hypoxia downregulates Beclin-1, a mediator of autophagy, in malignant intestinal epithelial cells. The reversal of this downregulation promoted autophagosome accumulation, enhanced the activation of a pro-apoptotic protease caspase-9 and subsequent caspase-9-dependent activation of two other pro-apoptotic proteases caspases 3 and 7 in these cells. Furthermore, the reversal of hypoxia-induced downregulation of Beclin-1 stimulated caspase-9-dependent apoptosis of the indicated cells under hypoxia. Interestingly, we found that Beclin-1-dependent caspase-9 activation in hypoxic cells was not associated with an increased release of cytochrome c from the mitochondria to the cytoplasm (such release represents a frequently occurring mechanism for caspase-9 activation). We also observed that Beclin-1-dependent apoptosis of hypoxic malignant cells was independent of FADD, a mediator of death receptor signaling. We conclude that hypoxia triggers a feedback mechanism that delays apoptosis of oxygen-deprived malignant intestinal epithelial cells and is driven by hypoxia-induced Beclin-1 downregulation. Thus, approaches aimed at the disruption of this mechanism can be expected to enhance the susceptibility of such cells to hypoxia-induced apoptosis.     

MicroRNA‐21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway

MicroRNAs and autophagy play critical roles in cardiac hypoxia/reoxygenation (H/R)‐induced injury. Here, we investigated the function of miR‐21 in regulating autophagy and identified the potential molecular mechanisms involved. To determine the role of miR‐21 in regulating autophagy, H9c2 cells were divided into the following six groups: control group, H/R group, (miR‐21+ H/R) group, (miR‐21‐negative control + H/R) group, (BEZ235+ H/R) group and (miR‐21+ BEZ235+ H/R) group. The cells underwent hypoxia for 1 hr and reoxygenation for 3 hrs. Cell count kit‐8 was used to evaluate cell function and apoptosis was analysed by Western blotting. Western blotting and transmission electron microscopy were used to investigate autophagy. We found that miR‐21 expression was down‐regulated, and autophagy was remarkably increased in H9c2 cells during H/R injury. Overexpression of miR‐21 with a miR‐21 precursor significantly inhibited autophagic activity and decreased apoptosis, accompanied by the activation of the AKT/mTOR pathway. In addition, treatment with BEZ235, a novel dual Akt/mTOR inhibitor, resulted in a significant increase in autophagy and apoptosis. However, we found that miR‐21‐mediated inhibition of apoptosis and autophagy was partly independent of Akt/mTOR activation, as demonstrated in cells treated with both miR‐21 and BEZ235. We showed that miR‐21 could inhibit H/R‐induced autophagy and apoptosis, which may be at least partially mediated by the Akt/mTOR signalling pathway.     

Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress‐induced autophagy

Autophagy is an evolutionary conserved process that degrades subcellular constituents. Unlike starvation‐induced autophagy, the molecular mechanism of genotoxic stress‐induced autophagy has not yet been fully elucidated. In this study, we analyze the molecular mechanism of genotoxic stress‐induced autophagy and identify an essential role of dephosphorylation of the Unc51‐like kinase 1 (Ulk1) at Ser⁶³⁷, which is catalyzed by the protein phosphatase 1D magnesium‐dependent delta isoform (PPM1D). We show that after exposure to genotoxic stress, PPM1D interacts with and dephosphorylates Ulk1 at Ser⁶³⁷ in a p53‐dependent manner. The PPM1D‐dependent Ulk1 dephosphorylation triggers Ulk1 puncta formation and induces autophagy. This happens not only in mouse embryonic fibroblasts but also in primary thymocytes, where the genetic ablation of PPM1D reduces the dephosphorylation of Ulk1 at Ser⁶³⁷, inhibits autophagy, and accelerates apoptosis induced by X‐ray irradiation. This acceleration of apoptosis is caused mainly by the inability of the autophagic machinery to degrade the proapoptotic molecule Noxa. These findings indicate that the PPM1D–Ulk1 axis plays a pivotal role in genotoxic stress‐induced autophagy.     

NLRX1/FUNDC1/NIPSNAP1‐2 axis regulates mitophagy and alleviates intestinal ischaemia/reperfusion injury

ObjectivesMitophagy is considered to be a key mechanism in the pathogenesis of intestinal ischaemic reperfusion (IR) injury. NOD‐like receptor X1 (NLRX1) is located in the mitochondria and is highly expressed in the intestine, and is known to modulate ROS production, mitochondrial damage, autophagy and apoptosis. However, the function of NLRX1 in intestinal IR injury is unclear.Materials and methodsNLRX1 in rats with IR injury or in IEC‐6 cells with hypoxia reoxygenation (HR) injury were measured by Western blotting, real‐time PCR and immunohistochemistry. The function of NLRX1‐FUNDC1‐NIPSNAP1/NIPSNAP2 axis in mitochondrial homeostasis and cell apoptosis were assessed in vitro.ResultsNLRX1 is significantly downregulated following intestinal IR injury. In vivo studies showed that rats overexpressing NLRX1 exhibited resistance against intestinal IR injury and mitochondrial dysfunction. These beneficial effects of NLRX1 overexpression were dependent on mitophagy activation. Functional studies showed that HR injury reduced NLRX1 expression, which promoted phosphorylation of FUN14 domain‐containing 1 (FUNDC1). Based on immunoprecipitation studies, it was evident that phosphorylated FUNDC1 could not interact with the mitophagy signalling proteins NIPSNAP1 and NIPSNAP2 on the outer membrane of damaged mitochondria, which failed to launch the mitophagy process, resulting in the accumulation of damaged mitochondria and epithelial apoptosis.ConclusionsNLRX1 regulates mitophagy via FUNDC1‐NIPSNAP1/NIPSNAP2 signalling pathway. Thus, this study provides a potential target for the development of a therapeutic strategy for intestinal IR injury.