AMPK mediates autophagy during myocardial ischemia in vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.     

The CD40-autophagy pathway is needed for host protection despite IFN-Γ-dependent immunity and CD40 induces autophagy via control of P21 levels

Autophagy degrades pathogens in vitro. The autophagy gene Atg5 has been reported to be required for IFN-γ-dependent host protection in vivo. However, these protective effects occur independently of autophagosome formation. Thus, the in vivo role of classic autophagy in protection conferred by adaptive immunity and how adaptive immunity triggers autophagy are incompletely understood. Employing biochemical, genetic and morphological studies, we found that CD40 upregulates the autophagy molecule Beclin 1 in microglia and triggers killing of Toxoplasma gondii dependent on the autophagy machinery. Infected CD40(-/-) mice failed to upregulate Beclin 1 in microglia/macrophages in vivo. Autophagy-deficient Beclin 1(+/-) mice, mice with deficiency of the autophagy protein Atg7 targeted to microglia/macrophages as well as CD40(-/-) mice exhibited impaired killing of T. gondii and were susceptible to cerebral and ocular toxoplasmosis. Susceptibility to toxoplasmosis occurred despite upregulation of IFN-γ, TNF-α and NOS2, preservation of IFN-γ-induced microglia/macrophage anti-T. gondii activity and the generation of anti-T. gondii T cell immunity. CD40 upregulated Beclin 1 and triggered killing of T. gondii by decreasing protein levels of p21, a molecule that degrades Beclin 1. These studies identified CD40-p21-Beclin 1 as a pathway by which adaptive immunity stimulates autophagy. In addition, they support that autophagy is a mechanism through which CD40-dependent immunity mediates in vivo protection and that the CD40-autophagic machinery is needed for host resistance despite IFN-γ.     

mTOR and Beclin1: Two key autophagy-related molecules and their roles in myocardial ischemia/reperfusion injury

Autophagy is the general term of lysosomal degradation of substances in cells, which is considered the key to maintaining the normal structure and function of the heart. It also has a correlation with several heart diseases, in particular, myocardial ischemia/reperfusion (I/R) injury. At the stage of myocardial ischemia, autophagy degrades nonfunctional cytoplasmic proteins providing the critical nutrients for the critical life activities, thereby suppressing cell apoptosis and necrosis. However, autophagy is likely to affect the heart negatively in the reperfusion stage. Mammalian target of rapamycin (mTOR) and Beclin1 are two vital autophagy-related molecules in myocardial I/R injury playing significant roles in different stages. In the ischemia stage, mTOR plays its roles through AMPK/mTOR and phosphoinositide 3-kinase/Akt/mTOR pathway, whereas Beclin1 plays its roles through its upregulation in the reperfusion stage. A possible interaction between mTOR and Beclin1 has been reported recently, and further studies need to be done to find the underlying interaction between the two molecules in myocardial I/R injury     

Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion

Autophagy is an intracellular bulk degradation process whereby cytoplasmic proteins and organelles are degraded and recycled through lysosomes. In the heart, autophagy plays a homeostatic role at basal levels, and the absence of autophagy causes cardiac dysfunction and the development of cardiomyopathy. Autophagy is induced during myocardial ischemia and further enhanced by reperfusion. Although induction of autophagy during the ischemic phase is protective, further enhancement of autophagy during the reperfusion phase may induce cell death and appears to be detrimental. In this review we discuss the functional significance of autophagy and the underlying signaling mechanism in the heart during ischemia/reperfusion.     

Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion

Autophagy is an intracellular bulk degradation process whereby cytoplasmic proteins and organelles are degraded and recycled through lysosomes. In the heart, autophagy plays a homeostatic role at basal levels, and the absence of autophagy causes cardiac dysfunction and the development of cardiomyopathy. Autophagy is induced during myocardial ischemia and further enhanced by reperfusion. Although induction of autophagy during the ischemic phase is protective, further enhancement of autophagy during the reperfusion phase may induce cell death and appears to be detrimental. In this review we discuss the functional significance of autophagy and the underlying signaling mechanism in the heart during ischemia/reperfusion.     

UVRAG: A New Player in Autophagy and Tumor Cell Growth

Autophagy has a well-documented role in the maintenance of homeostasis and the response to stressful environments and it is often deregulated in various human diseases including cancer. The regulation of the Beclin 1-PI3KC3 complex lipid kinase activity is a critical element in the autophagy signaling pathway. Previous studies1 have demonstrated that Beclin 1-PI3KC3-mediated autophagy is negatively regulated by a proto-oncogene Bcl-2. We have recently identified a novel coiled-coil UVRAG tumor suppressor candidate, which positively engages in Beclin 1-dependent autophagy. UVRAG interacts with Beclin 1, leading to activation of autophagy and thereof inhibition of tumorigenesis. This finding adds a new player to the emerging picture of the autophagy network, underscoring the importance of the coordinated activity between Bcl-2 and UVRAG in the regulation of Beclin 1-PI3KC3- mediated autophagy and tumor cell control.

Addendum to:

Autophagic and Tumor Suppressor Activity of a Novel Beclin 1-Binding Protein UVRAG

Chengyu Liang, Pinghui Feng, Bonsu Ku, Iris Dotan, Dan Canaani, Byung-Ha Oh and Jae U. Jung

Nature Cell Biol 2006; 8:688-99     

Neurodegeneration Induces Upregulation of Beclin 1

Autophagy, a bulk degradation of subcellular constituents, is activated in normal cell growth and development, and represents the major pathway by which the cell maintains a balance between protein synthesis and protein degradation. Autophagy was documented in several neurodegenerative diseases, and under stress conditions the autophagic process can lead to cell death (type II programmed cell death). Beclin 1 is a Bcl-2 interacting protein that was previously found to promote autophagy. We have used Beclin 1 protein as a marker for autophagy following traumatic brain injury in mice. We demonstrated a dramatic elevation in Beclin 1 levels near the injury site. Interestingly Beclin 1 elevation starts at early stages post injury (4 h) in neurons and 3 days later in astrocytes. In both cell types it lasts for at least three weeks. Neuronal cells, but not astrocytes, that overexpress Beclin 1 may exhibit damaged DNA but without changes in nuclear morphology. These observations may indicate that not all the Beclin 1 overexpressing cells will die. The elevation of Beclin 1 at the site of injury may represent enhanced autophagy as a mechanism to discard injured cells and reduce damage to cells by disposing of injured components.

Addenda to:

Closed Head Injury Induces Upregulation of Beclin 1 at the Cortical Site of Injury

T. Diskin, P. Tal-Or, S. Erlich, L. Mizrachy, A. Alexandrovich, E. Shohami and R. Pinkas-Kramarski

J Neurotrauma 2005; 22:750-62     

Zinc prevents mitochondrial superoxide generation by inducing mitophagy in the setting of hypoxia/reoxygenation in cardiac cells

Zinc plays a role in autophagy and protects cardiac cells from ischemia/reperfusion injury. This study aimed to test if zinc can induce mitophagy leading to attenuation of mitochondrial superoxide generation in the setting of hypoxia/reoxygenation (H/R) in cardiac cells. H9c2 cells were subjected to 4?h hypoxia followed by 2?h reoxygenation. Under normoxic conditions, treatments of cells with ZnCl₂ increased both the LC3-II/LC3-I ratio and GFP-LC3 puncta, implying that zinc induces autophagy. Further experiments showed that endogenous zinc is required for the autophagy induced by starvation and rapamycin. Zinc down-regulated TOM20, TIM23, and COX4 both in normoxic cells and the cells subjected to H/R, indicating that zinc can trigger mitophagy. Zinc increased ERK activity and Beclin1 expression, and zinc-induced mitophagy was inhibited by PD98059 and Beclin1 siRNA during reoxygenation. Zinc-induced Beclin1 expression was reversed by PD98059, implying that zinc promotes Beclin1 expression via ERK. In addition, zinc failed to induce mitophagy in cells transfected with PINK1 siRNA and stabilized PINK1 in mitochondria. Moreover, zinc-induced PINK1 stabilization was inhibited by PD98059. Finally, zinc prevented mitochondrial superoxide generation and dissipation of mitochondrial membrane potential (ΔΨ_m) at reoxygenation, which was blocked by both the Beclin1 and PINK1 siRNAs, suggesting that zinc prevents mitochondrial oxidative stress through mitophagy. In summary, zinc induces mitophagy through PINK1 and Beclin1 via ERK leading to the prevention of mitochondrial superoxide generation in the setting of H/R. Clearance of damaged mitochondria may account for the cardioprotective effect of zinc on H/R injury.     

A Novel Role for Autophagy in Neurodevelopment

We recently showed that Ambra1, a WD40-containing ~130 KDa protein, is a novel activating molecule in Beclin 1-regulated autophagy and plays a role in the development of the nervous system. Ambra1 binds to Beclin 1 and favors Beclin 1/Vps34 interaction. At variance with these factors, Ambra1 is highly conserved among vertebrates only, and its expression is mostly confined to the neuroepithelium during early neurogenesis. Ambra1 functional inactivation in mouse led to lethality in utero (starting from embryonic day 14.5), characterized by severe neural tube defects associated with autophagy impairment, unbalanced cell proliferation, accumulation of ubiquitinated proteins, and excessive apoptosis. We also demonstrated that hyperproliferation was the earliest detectable abnormality in the developing neuroepithelium, followed by a wave of caspase-dependent cell death. These findings provided in vivo evidence supporting the existence of a complex interplay between autophagy, cell proliferation and cell death during neural development in mammals. In this Addendum, we review our findings in the contexts of autophagy and neurodevelopment and consider some of the issues raised.

Addendum to:

Ambra1 Regulates Autophagy and the Development of the Nervous System

G.M. Fimia, A. Stoykova, A. Romagnoli, L. Giunta, S. Di Bartolomeo, R. Nardacci, M. Corazzari, C. Fuoco, A. Ucar, P. Schwartz, P. Gruss, M. Piacentini, K. Chowdhury and F. Cecconi

Nature 2007; In press     

Protective Role of AMP-Activated Protein Kinase-Evoked Autophagy on an In Vitro Model of Ischemia/Reperfusion-Induced Renal Tubular Cell Injury

Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs such as brain, heart, and kidneys. Renal injury from I/R, which may occur in renal transplantation, surgery, trauma, or sepsis, is known to be an important cause of acute kidney injury. The detailed molecular mechanism of renal I/R injury is still not fully clear. Here, we investigate the role of AMP-activated protein kinase (AMPK)-evoked autophagy in the renal proximal tubular cell death in an in vitro I/R injury model. To mimic in vivo renal I/R injury, LLC-PK1 cells, a renal tubular cell line derived from pig kidney, were treated with antimycin A and 2-deoxyglucose to mimic ischemia injury followed by reperfusion with growth medium. This I/R injury model markedly induced apoptosis and autophagy in LLC-PK1 cells in a time-dependent manner. Autophagy inhibitor 3-methyladenine (3MA) significantly enhanced I/R injury-induced apoptosis. I/R could also up-regulate the phosphorylation of AMPK and down-regulate the phosphorylation of mammalian target of rapamycin (mTOR). Cells transfected with small hairpin RNA (shRNA) for AMPK significantly increased the phosphorylation of mTOR as well as decreased the induction of autophagy followed by enhancing cell apoptosis during I/R. Moreover, the mTOR inhibitor RAD001 significantly enhanced autophagy and attenuated cell apoptosis during I/R. Taken together, these findings suggest that autophagy induction protects renal tubular cell injury via an AMPK-regulated mTOR pathway in an in vitro I/R injury model. AMPK-evoked autophagy may be as a potential target for therapeutic intervention in I/R renal injury.     

Role of Autophagy in Breast Cancer

Autophagy is an evolutionarily conserved process of cytoplasm and cellular organelle degradation in lysosomes. Autophagy is a survival pathway required for cellular viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. In neurons, constitutive autophagy limits accumulation of polyubiquitinated proteins and prevents neuronal degeneration. Therefore, autophagy has emerged as a homeostatic mechanism regulating the turnover of long-lived or damaged proteins and organelles, and buffering metabolic stress under conditions of nutrient deprivation by recycling intracellular constituents. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin1^+/- mice are tumor-prone. We found that allelic loss of beclin1 renders immortalized mouse mammary epithelial cells susceptible to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. Thus, loss of the prosurvival role of autophagy likely contributes to breast cancer progression by promoting genome damage and instability. Exploring the yet unknown relationship between defective autophagy and other breast cancer-promoting functions may provide valuable insight into the pathogenesis of breast cancer and may have significant prognostic and therapeutic implications for breast cancer patients.

Addendum to:

Autophagy Mitigates Metabolic Stress and Genome Damage in Mammary Tumorigenesis

V. Karantza-Wadsworth, S. Patel, O. Kravchuk, G. Chen, R. Mathew, S. Jin and E. White

Genes Dev 2007; 21:1621-35     

Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.     

Autophagy decreases alveolar epithelial cell injury by regulating the release of inflammatory mediators

To research the impact of autophagy on alveolar epithelial cell inflammation and its possible mechanism in the early stages of hypoxia, we established a cell hypoxia–reoxygenation model and orthotopic left lung ischemia–reperfusion model. Rat alveolar epithelial cells stably expressing GFP-LC3 were treated with an autophagy inhibitor (3-MA) or an autophagy promoter (rapamycin), followed by hypoxia–reoxygenation treatment for 2, 4, and 6 hr in vitro. In vivo, 20 male Sprague Dawley rats were randomly divided into four groups (model group: No blocking of the hilum in the left lung; control group: Blocking of the hilum in the left lung for 1 hr with dimethyl sulfoxide lavage; 3-MA group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of 3-MA (5 μmol/L) solution lavage; and rapamycin group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of rapamycin (250 nmol/L) solution lavage) to establish an orthotopic left lung ischemia model. This study demonstrated that rapamycin significantly suppressed the nuclear factor kappa B signaling pathway and limited the expression of proinflammatory factors. A contrary result was found after the 3-MA pretreatment. These findings indicate that autophagy reduces ischemia–reperfusion injury by repressing inflammatory signaling pathways in the early stages of hypoxia in vitro and in vivo. Autophagy could be a new protective method for application in lung ischemia–reperfusion injury.     

PKCδ and Tissue Transglutaminase are Novel Inhibitors of Autophagy in Pancreatic Cancer Cells

Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKCδ) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKCδ/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKCδ/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.

Addendum to:

Tissue Transglutaminase Inhibits Autophagy in Pancreatic Cancer Cells

U. Akar, B. Ozpolat, K. Mehta, J. Fok, Y. Kondo and G. Lopez-Berestein

Mol Cancer Res 2007; 5:241-9     

Lactoferrin promotes the autophagy activity during osteoblast formation via BCL2-Beclin1 signaling

Background

Lactoferrin, as the main component of milk, can maintain osteoblast formation, which is conducive to the prevention and treatment of osteoporosis. Lactoferrin also serves as an autophagy regulator, especially in osteoblasts. This study aimed to explore the significance of autophagy in osteoblast formation regulated by lactoferrin and the internal mechanism.

Methods and results

In this study, we firstly explored the roles of lactoferrin in the autophagy activity of primary osteoblasts (LC3 transformation rate, autophagosome formation). Subsequently, we further investigated the effects of lactoferrin on the BCL2 expression and BCL2-Beclin1 complex. Ultimately, the significance of BCL2 overexpression and Beclin1 silencing on lactoferrin-regulated osteoblast autophagy and osteogenic parameters (ALP activity and mRNA expression of PCNA, Col1, BGLAP and OPN) was observed by gene processing, respectively. Our results showed that lactoferrin enhanced the autophagy activity of osteoblasts. Importantly, lactoferrin inhibited BCL2 expression and the co-immunoprecipitation of BCL2 and Beclin1 in osteoblasts. Moreover, lactoferrin-promoted autophagy and osteogenic parameters was reversed by BCL2 overexpression or Beclin1 silencing in osteoblasts.

Conclusions

In conclusion, lactoferrin can inhibit BCL2 expression in osteoblasts, further enhancing Beclin1-dependent autophagy activation.

     

VDAC1 promotes cardiomyocyte autophagy in anoxia/reoxygenation injury via the PINK1/Parkin pathway

Ischemia/reperfusion (I/R) is a well-known injury to the myocardium, but the mechanism involved remains elusive. In addition to the well-accepted apoptosis theory, autophagy was recently found to be involved in the process, exerting a dual role as protection in ischemia and detriment in reperfusion. Activation of autophagy is mediated by mitochondrial permeability transition pore (MPTP) opening during reperfusion. In our previous study, we showed that MPTP opening is regulated by VDAC1, a channel protein located in the outer membrane of mitochondria. Thus, upregulation of VDAC1 expression is a possible trigger to cardiomyocyte autophagy via an unclear pathway. Here, we established an anoxia/reoxygenation (A/R) model in vitro to simulate the I/R process in vivo. At the end of A/R treatment, VDAC1, Beclin 1, and LC3-II/I were upregulated, and autophagic vacuoles were increased in cardiomyocytes, which showed a connection of VDAC1 and autophagy development. These variations also led to ROS burst, mitochondrial dysfunction, and aggravated apoptosis. Knockdown of VDAC1 by RNAi could alleviate the above-mentioned cellular damages. Additionally, the expression of PINK1 and Parkin was enhanced after A/R injury. Furthermore, Parkin was recruited to mitochondria from the cytosol, which suggested that the PINK1/Parkin autophagic pathway was activated during A/R. Nevertheless, the PINK1/Parkin pathway was effectively inhibited when VDAC1 was knocked-down. Taken together, the A/R-induced cardiomyocyte injury was mediated by VDAC1 upregulation, which led to cell autophagy via the PINK1/Parkin pathway, and finally aggravated apoptosis.     

细胞自噬在大鼠缺血/再灌注肺损伤中的调控作用

目的: 探讨细胞自噬在大鼠缺血/再灌注肺损伤中的作用。方法: 随机将40只SD大鼠分为5组(n=8),分别为 ① 假手术组(Sham组):只开胸3.5 h;② 缺血/再灌注组(I/R组):开胸夹闭肺门缺血0.5 h后再灌注3 h;③ 溶剂组(DMSO组):术前1 h腹腔注射DMSO溶液;④自噬激动剂组(Rap组):术前腹腔注射雷帕霉素溶液;⑤自噬抑制剂组(3-MA组):术前1 h腹腔注射3-MA溶液;后三组的其余操作同I/R组。实验结束后处死大鼠,取肺组织,记录并计算肺组织湿/干重比(W/D)、总肺含水量变化(TLW) ,光镜和电镜观察肺组织及细胞形态,计算肺泡损伤率(IAR),Western blot检测自噬相关蛋白的表达情况。结果: 相对于sham组,其余四组肺W/D、TLW、IAR均明显升高,自噬相关蛋白表达明显上升,p-AMPK、Beclin 1、LC3 II 蛋白明显增多,p-mTOR、p62蛋白明显减少(P＜0.05或P＜0.01),光镜下其余各组肺组织有不同程度的水肿渗出,肺泡结构紊乱,电镜下细胞超微结构损伤加重,部分可见自噬小体;与DMSO组相比,3-MA组肺W/D、TLW、IAR明显下降(P＜0.05或P＜0.01),自噬相关蛋白表达明显下降,肺间质水肿较轻,细胞渗出较少,细胞超微结构损伤减轻,未见自噬小体。而I/R、DMSO、Rap组的各项指标变化无统计学差异(P＞0.05)。结论: 肺缺血/再灌注可诱发细胞自噬增强,从而引起大鼠肺损伤。     

Erratum to: Sinha S,Colbert CL,Becker N,Wei Y,Levine B. Molecular basis of the regulation of Beclin 1-dependent autophagy by the γ-herpesvirus 68 Bcl-2 homolog M11. Autophagy 2008; 4:989-97

Table 1 in Molecular basis of the regulation of Beclin 1-dependent autophagy by the γ-herpesvirus 68 Bcl-2 homolog M11 (Autophagy 2008; 4:989-97) contained an error in the heading of the last column. "BAD" should have appeared as "BAX." This error has been corrected and the corrected paper can now be downloaded at: http://www.landesbioscience.com/journals/3/article/6803.     

TRIM50 regulates Beclin 1 proautophagic activity

Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation.