Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells.

Autophagy is required for benign hepatic tumors to progress into malignant hepatocellular carcinoma. However, the mechanism is unclear. Here, we report that mitophagy, the selective removal of mitochondria by autophagy, positively regulates hepatic cancer stem cells (CSCs) by suppressing the tumor suppressor p53. When mitophagy is enhanced, p53 co-localizes with mitochondria and is removed by a mitophagy-dependent manner. However, when mitophagy is inhibited, p53 is phosphorylated at serine-392 by PINK1, a kinase associated with mitophagy, on mitochondria and translocated into the nucleus, where it binds to the NANOG promoter to prevent OCT4 and SOX2 transcription factors from activating the expression of NANOG, a transcription factor critical for maintaining the stemness and the self-renewal ability of CSCs, resulting in the reduction of hepatic CSC populations. These results demonstrate that mitophagy controls the activities of p53 to maintain hepatic CSCs and provide an explanation as to why autophagy is required to promote hepatocarcinogenesis.

TNF-α Induced by Hepatitis C Virus via TLR7 and TLR8 in Hepatocytes Supports Interferon Signaling via an Autocrine Mechanism.

Invasion by infectious pathogens can elicit a range of cytokine responses from host cells. These cytokines provide the initial host defense mechanism. In this report, we demonstrate that TNF-α, a pro-inflammatory cytokine, can be induced by hepatitis C virus (HCV) in its host cells in a biphasic manner. The initial induction of TNF-α by HCV was prompt and could be blocked by the antibody directed against the HCV E2 envelope protein and by chemicals that inhibit endocytosis, indicating the specificity of endocytic uptake of HCV in this induction. Further studies indicated that the induction of TNF-α was dependent on toll-like receptors 7 and 8 (TLR7/8) but not on other intracellular pattern recognition receptors. Consistently, siRNA-mediated gene silencing of the downstream effectors in the TLR7/8 signaling pathway including MyD88, IRAK1, TRAF6, TAK1 and p65 NF-κB suppressed the expression of TNF-α. The role of p65 NF-κB in the induction of TNF-α via transcriptional up-regulation was further confirmed by the chromatin immunoprecipitation assay. TNF-α induced by HCV could activate its own receptor TNFR1 on hepatocytes to suppress HCV replication. This suppressive effect of TNF-α on HCV was due to its role in supporting interferon signaling, as the suppression of its expression led to the loss of IFNAR2 and impaired interferon signaling and the induction of interferon-stimulated genes. In conclusion, our results indicate that hepatocytes can sense HCV infection via TLR7/8 to induce the expression of TNF-α, which inhibits HCV replication via an autocrine mechanism to support interferon signaling.

Impaired lung homeostasis in neonatal mice exposed to cigarette smoke.

In infants, smoke exposure is associated with more respiratory illnesses and decreased lung function. We hypothesized that perinatal lung is particularly susceptible to the damaging effects of cigarette smoke (CS) and that exposure to CS during this period may alter expression of immune response genes and adversely affect lung growth. To test this, we exposed neonatal mice to 14 days of CS. Immediately after exposure to CS, pulmonary gene expression profiling was performed on 2-week-old CS-exposed lung and age-matched control lung. Nitrotyrosine, TUNEL, MAC3, and phospho-SMAD-2 (p-SMAD2) staining was also performed. At 8 weeks of age, lung volume measurements were determined and mean linear intercept measurements were calculated. Pulmonary gene expression profiling revealed that CS exposure significantly inhibited type 1 and type 2 interferon pathway genes in neonatal lung, compared with age-matched control lung. Neonatal CS-exposed lung also had a significant increase in n-tyrosine, TUNEL, and p-SMAD2 staining when compared with adult CS-exposed lung and age-matched control lung. Lung volumes at 8 weeks of age were modestly but significantly decreased in mice exposed to CS in the neonatal period compared with age-matched controls, consistent with impaired lung growth. The results of this study indicate that exposure to CS during the neonatal period inhibits expression of genes involved in innate immunity and mildly impairs postnatal lung growth. These findings may in part explain the increased incidence of respiratory symptoms in infants and children exposed to CS.

IL-12 overexpression in mice as a model for Sjögren lung disease.

Interleukin-12 (IL-12), a Th1 proinflammatory cytokine, is reported to be increased in Sjögren syndrome. To evaluate the effects of local Th1/Th2 deregulation, we generated a transgenic mouse model that overexpresses IL-12 in the lungs. IL-12 transgenic mice developed bronchial and alveolar abnormalities strikingly similar to those found in the lungs of Sjögren patients. Pathologically, lung abnormalities began at approximately 4 mo of age and were characterized by lymphocytic infiltrates around the bronchi, intraluminal periodic acid Schiff-positive debris, increased cell proliferation in the alveolar region, and increased interstitial and alveolar macrophages. Functionally, these abnormalities translated into decreased mucociliary clearance (P<0.05 vs. wild-type littermates) and increased oxidative stress (P<0.01). The pathological and functional abnormalities were accompanied by significant changes in lung natural killer (NK) cells. The number of NK cells was fourfold higher in IL-12 transgenic than wild-type lungs (20% of all lymphoid cells vs. 5%) during the first month of life. NK cells then decreased within a narrow window of time (from 30 to 50 days of age), reaching a nadir of approximately 2% on day 50, and remained at these low levels thereafter. This new mouse model highlights the role of IL-12 in the initiation of Sjögren syndrome.

Vascular endothelial growth factor receptor 2 blockade disrupts postnatal lung development.

Vascular endothelial growth factor (VEGF) is necessary for normal postnatal lung development and may underlie the structural lung damage that follows hyperoxic exposure. To determine the individual roles of VEGF receptors (VEGFR) 2 and 1 on postnatal lung growth, neonatal mice were treated with neutralizing antibodies to VEGFR-2 (DC101) or VEGFR-1 (MF1) in the perinatal period. At 1 wk of age, mice treated with DC101 on Days 2 and 4 of life had significantly larger mean alveolar diameters consistent with impaired alveolization. By 2 wk of age, however, perinatally treated DC101 mice had normal-appearing alveolar structure. Mice exposed to perinatal hyperoxia (O(2)) also had larger mean alveolar diameters at 1 wk of age, but unlike DC101-treated mice, their mitotic index was decreased at 1 wk of age and they had persistent alveolar enlargement beyond the first 2 wk of life. The O(2)-treated lung also had an increase in caspase 3 at 1 wk of age and significantly greater expression of nitrotyrosine at 2 wk of age. Therefore, VEGFR-2 blockade in the perinatal period disrupts early alveolar development, but the effect is reversible with time, whereas hyperoxic lung injury is associated with ongoing lung structural impairment.

The effect of glutamine on A549 cells exposed to moderate hyperoxia.

The use of high oxygen concentrations is frequently necessary in the treatment of acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD). High oxygen concentrations, however, are detrimental to cell growth and cell survival. Glutamine (Gln) may be protective to cells during periods of stress and recently has been shown to increase survival in A549 cells exposed to lethal concentrations of oxygen (95% O2). We found that supplemental Gln enhances cell growth in A549 cells exposed to moderate concentrations of oxygen (60% O2). We therefore evaluated the effect of moderate hyperoxia on the cell cycle distribution of A549 cells. At 48 h there was no significant difference in the cell cycle distribution between 2 mM Gln cells in 60% O2 and 2 mM cells in room air. Furthermore, 2 mM Gln cells in 60% O2 had stable protein levels of cyclin B1 consistent with ongoing cell proliferation. In contrast, at 48 h, cells not supplemented with glutamine (Gln-) in 60% O2 had evidence of growth arrest by both flow cytometry (increased percentage of G1 cells) and by decreased protein levels of cyclin B1. G1 growth arrest in the Gln- cells exposed to 60% O2 was not, however, associated with induction of p21 protein. At 72 and 96 h, Gln- cells in 60% O2, began to demonstrate a partial loss of G1 checkpoint regulation and an increase in apoptosis, indicating an increased sensitivity to oxygen toxicity. Glutathione (GSH) concentrations were then measured. 2 mM Gln cells in 60% O2 were found to have higher concentrations of GSH compared to Gln- cells in 60% O2, suggesting that Gln confers protection to the cell during exposure to hyperoxia through up-regulation of GSH. When cells in 60% O2 were given higher concentrations of Gln (5 and 10 mM), cell growth at 96 h was increased compared to cells grown in 2 mM Gln (P<0.04). Clonal survival was also increased in cells exposed 60% O2 and supplemented with higher concentrations of Gln compared to Gln- cells in 60% O2. These studies suggest that supplemental glutamine may improve cell growth and cell viability and therefore may be beneficial to the lung during exposure to moderate concentrations of supplemental oxygen.

The effect of neonatal hyperoxia on the lung of p21Waf1/Cip1/Sdi1-deficient mice.

Hyperoxia is an important factor in the development of bronchopulmonary dysplasia and is associated with growth arrest and impaired alveolar septal development in the neonatal lung. p21(Waf1/Cip1/Sdi1) (p21), a cyclin-dependent kinase inhibitor, acts as a checkpoint regulator in the cell cycle during periods of stress and is induced in neonatal lung during hyperoxia exposure. To determine if p21 protects against lung injury during neonatal lung development, we placed newborn p21 knockout (p21(-/-)) and p21 wild-type (p21(+/+)) mice in 85-90% O(2) for 4 d. We found that newborn p21(-/-) mice exposed to O(2) had decreased survival in hyperoxia compared with p21(+/+) mice (P < 0.01). At 2 and 6 wk after exposure to neonatal hyperoxia, p21(-/-) O(2) lung had significantly larger alveoli then p21(-/-) control lung, as assessed by mean alveolar size and mean linear intercept. Pulmonary function tests at 6 wk demonstrated increased lung volume in both p21(-/-) and p21(+/+) O(2) mice consistent with altered lung growth from neonatal exposure to hyperoxia. Antibodies to nitrotyrosine, a marker for oxidative stress revealed that at 2 and 6 wk of age, p21(-/-) O(2) lung had significantly more oxidative stress than p21(-/-) and p21(+/+) control and p21(+/+) O(2) lung. We therefore conclude that p21 confers some additional protection to the lung during exposure to neonatal hyperoxia. Furthermore, p21 may be important during recovery from lung injury because it is associated with lower levels of oxidative stress and increased oxidative stress may contribute to alveolar growth abnormalities in the p21(-/-) O(2) lung.