Up-regulation of autophagy in small intestine Paneth cells in response to total-body γ-irradiation

Armed Forces Radiobiology Research Institute, Bethesda, MD 20889-5603, USA.
The Journal of Pathology (Impact Factor: 7.43). 10/2009; 219(2):242-52. DOI: 10.1002/path.2591
Source: PubMed


Macroautophagy (mAG) is a lysosomal mechanism of degradation of cell self-constituents damaged due to variety of stress factors, including ionizing irradiation. Activation of mAG requires expression of mAG protein Atg8 (LC3) and conversion of its form I (LC3-I) to form II (LC3-II), mediated by redox-sensitive Atg4 protease. We have demonstrated upregulation of this pathway in the innate host defense Paneth cells of the small intestine (SI) due to ionizing irradiation and correlation of this effect with induction of pro-oxidant inducible nitric oxide synthase (iNOS). CD2F1 mice were exposed to 9.25 Gy gamma-ionizing irradiation. Small intestinal specimens were collected during 7 days after ionizing irradiation. Assessment of ionizing irradiation-associated alterations in small intestinal crypt and villus cells and activation of the mAG pathway was conducted using microscopical and biochemical techniques. Analysis of iNOS protein and the associated formation of nitrites and lipid peroxidation products was performed using immunoblotting and biochemical analysis, and revealed increases in iNOS protein, nitrate levels and oxidative stress at day 1 following ionizing irradiation. Increase in immunoreactivity of LC3 protein in the crypt cells was observed at day 7 following ionizing irradiation. This effect predominantly occurred in the CD15-positive Paneth cells and was associated with accumulation of LC3-II isoform. The formation of autophagosomes in Paneth cells was confirmed by transmission electron microscopy (TEM). Up-regulation of LC3 pathway in the irradiated SI was accompanied by a decreased protein-protein interaction between LC3 and chaperone heat shock protein 70. A high-level of LC3-immunoreactivity in vacuole-shaped structures was spatially co-localized with immunoreactivity of 3-nitro-tyrosine. The observed effects were diminished in iNOS knockout B6.129P2-NOS2(tm1Lau)/J mice subjected to the same treatments. We postulate that the observed up-regulation of mAG in the irradiated small intestine is at least in part mediated by the iNOS signalling mechanism.

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Available from: Nikolai Gorbunov
    • "Among the key molecules activated during radiation exposure, inducible nitric oxide synthase gene (iNOS) and nitric oxide (NO) have been shown to be involved in radiation induced apoptosis and autophagy[43,44]. As iNOS gene promotor region contains motifs of many transcription factors such as nuclear factor iB (NF-iB) and kruppel like factor 6 (KLF6), it results in increased NO production that causes caspase-mediated apoptosis and protein nitration-mediated autophagic induc- tion[43]. Radiation-induced oxidative stress can cause compromised mitochondrial functioning, protein misfolding and ER stress, besides DNA damage. "
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    ABSTRACT: Autophagy is an evolutionary conserved, indispensable, lysosome-mediated degradation process, which helps in maintaining homeostasis during various cellular traumas. During stress, a context-dependent role of autophagy has been observed which drives the cell towards survival or death depending upon the type, time, and extent of the damage. The process of autophagy is stimulated during various cellular insults, e.g. oxidative stress, endoplasmic reticulum stress, imbalances in calcium homeostasis, and altered mitochondrial potential. Ionizing radiation causes ROS-dependent as well as ROS-independent damage in cells that involve macromolecular (mainly DNA) damage, as well as ER stress induction, both capable of inducing autophagy. This review summarizes the current understanding on the roles of oxidative stress, ER stress, DNA damage, altered mitochondrial potential, and calcium imbalance in radiation-induced autophagy as well as the merits and limitations of targeting autophagy as an approach for radioprotection and radiosensitization.
    No preview · Article · Jan 2016 · Free Radical Research
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    • "For normal tissues, there are only sporadic data on how IR affects autophagy at therapeutic doses. Gorbunov et al. reported an autophagy increase in the crypt cells of the murine small intestine 7 days after the exposure to γ-radiation [17]. "
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    ABSTRACT: Objectives Vasculature damage is an important contributor to the side-effects of radiotherapy. The aim of this study is to provide insights into the radiobiology of the autophagic response of endothelial cells. Methods and Materials Human umbilical vascular endothelial cells (HUVEC) were exposed to 2 Gy of ionizing radiation (IR) and studied using confocal microscopy and western blot analysis, at 4 and 8 days post-irradiation. The role of autophagy flux in HUVEC radio-sensitivity was also examined. Results IR-induced accumulation of LC3A+, LC3B+ and p62 cytoplasmic vacuoles, while in double immunostaining with lysosomal markers (LAMP2a and CathepsinD) repression of the autophagolysosomal flux was evident. Autophagy-related proteins (ATF4, HIF1α., HIF2α, Beclin1) were, however, induced excluding an eventual repressive effect of radiation on autophagy initiating protein expression. Exposure of HUVEC to SMER28, an mTOR-independent inducer of autophagy, enhanced proLC3 and LC3A, B-I protein expression and accelerated the autophagic flux. Pre-treatment of HUVEC with SMER28 protected against the blockage of autophagic flux induced by IR and conferred radio-resistance. Suppression of LC3A/LC3B proteins with siRNAs resulted in radio-sensitization. Conclusions The current data provide a rationale for the development of novel radioprotection policies targeting the autophagic pathway.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "The misregulation of the autolysosomal pathway during autophagy can eventually cause cell death either by triggering apoptosis in apoptosis-sensitive cells or as a result of destructive self-digestion [38]. Light chain 3 (LC3) is a protein involved in the formation of autophagosomes in mammalian cells that serves as a biomarker for occurrence of autophagy [13]. "

    Full-text · Dataset · Jun 2014
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