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,
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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.
    PLoS ONE 07/2014; 9(7):e102408. DOI:10.1371/journal.pone.0102408 · 3.23 Impact Factor
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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]. "
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    • "CIP treatment also significantly reduced the number of cells that underwent CI-induced autophagy indicated by the LC3 presence in crypt cells (Figure 6B; green). In contrast to apoptosis seen in villous epithelium, autophagic cells were found only in crypt cells as previously described [18]. "
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    ABSTRACT: Radiation combined injury (CI) is a radiation injury (RI) combined with other types of injury, which generally leads to greater mortality than RI alone. A spectrum of specific, time-dependent pathophysiological changes is associated with CI. Of these changes, the massive release of pro-inflammatory cytokines, severe hematopoietic and gastrointestinal losses and bacterial sepsis are important treatment targets to improve survival. Ciprofloxacin (CIP) is known to have immunomodulatory effect besides the antimicrobial activity. The present study reports that CIP ameliorated pathophysiological changes unique to CI that later led to major mortality. B6D2F1/J mice received CI on day 0, by RI followed by wound trauma, and were treated with CIP (90 mg/kg p.o., q.d. within 2 h after CI through day 10). At day 10, CIP treatment not only significantly reduced pro-inflammatory cytokine and chemokine concentrations, including interleukin-6 (IL-6) and KC (i.e., IL-8 in human), but it also enhanced IL-3 production compared to vehicle-treated controls. Mice treated with CIP displayed a greater repopulation of bone marrow cells. CIP also limited CI-induced apoptosis and autophagy in ileal villi, systemic bacterial infection, and IgA production. CIP treatment led to LD0/10 compared to LD20/10 for vehicle-treated group after CI. Given the multiple beneficial activities of CIP shown in our experiments, CIP may prove to be a useful therapeutic drug for CI.
    PLoS ONE 03/2013; 8(3):e58389. DOI:10.1371/journal.pone.0058389 · 3.23 Impact Factor
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