Sestrins (Sesns) are a family of highly conserved stress-responsive proteins, transcriptionally regulated by p53 and forkhead transcription factor that exhibit oxidoreductase activity in vitro and can protect cells from oxidative stress. However, their major biochemical and physiological function does not appear to depend on their redox (reduction and oxidation) activity. Sesns promote activation of adenosine-5'-monophosphate (AMP)-dependent protein kinase in both mammals and flies. Stress-induced Sesn expression results in inhibition of the target of rapamycin complex 1 (TORC1) and the physiological and pathological implications of disrupting the Sesns-TORC1 crosstalk are now being unravelled. Detailing their mechanism of action and exploring their roles in human physiology point to exciting new insights to topics as diverse as stress, cancer, metabolism and aging.

Vascular endothelium provides a selective barrier between the blood and tissues, participates in wound healing and angiogenesis, and regulates tissue recruitment of inflammatory cells. Nuclear factor (NF)-κB transcription factors are pivotal regulators of survival and inflammation, and have been suggested as potential therapeutic targets in cancer and inflammatory diseases. Here we show that mice lacking IKKβ, the primary kinase mediating NF-κB activation, are smaller than littermates and born at less than the expected Mendelian frequency in association with hypotrophic and hypovascular placentae. IKKβ-deleted endothelium manifests increased vascular permeability and reduced migration. Surprisingly, we find that these defects result from loss of kinase-independent effects of IKKβ on activation of the serine-threonine kinase, Akt. Together, these data demonstrate essential roles for IKKβ in regulating endothelial permeability and migration, as well as an unanticipated connection between IKKβ and Akt signalling.

Hepatocyte I kappaB kinase beta (IKK beta) inhibits hepatocarcinogenesis by suppressing accumulation of reactive oxygen species (ROS) and liver damage, whereas JNK1 activation promotes ROS accumulation, liver damage, and carcinogenesis. We examined whether hepatocyte p38 alpha, found to inhibit liver carcinogenesis, acts similarly to IKK beta in control of ROS metabolism and cell death. Hepatocyte-specific p38 alpha ablation enhanced ROS accumulation and liver damage, which were prevented upon administration of an antioxidant. In addition to elevated ROS accumulation, hepatocyte death, augmented by loss of either IKK beta or p38 alpha, was associated with release of IL-1 alpha. Inhibition of IL-1 alpha action or ablation of its receptor inhibited carcinogen-induced compensatory proliferation and liver tumorigenesis. IL-1 alpha release by necrotic hepatocytes is therefore an important mediator of liver tumorigenesis.

We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

Although stem cell-mediated treatment of ischemic diseases offers significant therapeutic promise, the limitation in the therapeutic efficacy of transplanted stem cells in vivo because of poor engraftment remains a challenge. Several strategies aimed at improving survival and engraftment of stem cells in the ischemic myocardium have been developed, such as cell transplantation in combination with growth factor delivery, genetic modification of stem cells, and/or cell therapy using scaffolds. To improve therapeutic efficacy, we investigated the effects of genistein on the engraftment of transplanted ECFCs in an acute myocardial ischemia model.

Although mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine, ischemia-induced endoplasmic reticulum (ER) stress induces low MSC engraftment and limits their therapeutic efficacy. To overcome this, we investigated the protective effect of tauroursodeoxycholic acid (TUDCA), a bile acid, on ER stress in MSCs in vitro and in vivo. In ER stress conditions, TUDCA treatment of MSCs reduced the activation of ER stress-associated proteins, including GRP78, PERK, eIF2α, ATF4, IRE1α, JNK, p38, and CHOP. In particular, TUDCA inhibited the dissociation between GRP78 and PERK, resulting in reduced ER stress-mediated cell death. Next, to explore the ER stress protective mechanism induced by TUDCA treatment, TUDCA-mediated cellular prion protein (PrPC) activation was assessed. TUDCA treatment increased PrPC expression, which was regulated by Akt phosphorylation. Manganese-dependent superoxide dismutase (MnSOD) expression also increased significantly in response to signaling through the TUDCA-Akt axis. In a murine hindlimb ischemia model, TUDCA-treated MSC transplantation augmented the blood perfusion ratio, vessel formation, and transplanted cell survival more than untreated MSC transplantation did. Augmented functional recovery following MSC transplantation was blocked by PrPC downregulation. This study is the first to demonstrate that TUDCA protects MSCs against ER stress via Akt-dependent PrPC and Akt-MnSOD pathway.

Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk (-/-) MSCs. An ex vivo adipogenic differentiation assay showed that Lnk (-/-) MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R-Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk (-/-) MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway.

Toll-like Receptor 3 (TLR3) is a pathogen pattern recognition receptor that plays a key role in innate immunity. TLR3 signalling has numerous functions in liver, both in health and disease. Here we report that TLR3 is expressed by quiescent hepatic stellate cells (HSC) where it functions to induce transcription and secretion of functional interferons as well as a number of other cytokines and chemokines. Upon transdifferentiation into myofibroblasts, HSCs rapidly loose the ability to produce interferon gamma (IFNγ). Mechanistically, this gene silencing may be due to Polycomb complex mediated repression via methylation of histone H3 lysine 27. In contrast to wild type, quiescent HSC isolated from tlr3 knockout mice do not produce IFNγ in response to Poly(I∶C) treatment. Therefore, quiescent HSC may contribute to induction of the hepatic innate immune system in response to injury or infection.

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc7-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc7-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc7-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc7-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.

Exposure to hepatitis C virus (HCV) typically results in chronic infection that leads to progressive liver disease ranging from mild inflammation to severe fibrosis and cirrhosis as well as primary liver cancer. HCV triggers innate immune signaling within the infected hepatocyte, a first step in mounting of the adaptive response against HCV infection. Persistent inflammation is strongly associated with liver tumorigenesis. The goal of our work was to investigate the initiation of the inflammatory processes triggered by HCV viral proteins in their host cell and their possible link with HCV-related liver cancer. We report a dramatic upregulation of the lymphotoxin signaling pathway and more specifically of lymphotoxin-β in tumors of the FL-N/35 HCV-transgenic mice. Lymphotoxin expression is accompanied by activation of NF-κB, neosynthesis of chemokines and intra-tumoral recruitment of mononuclear cells. Spectacularly, IKKβ inactivation in FL-N/35 mice drastically reduces tumor incidence. Activation of lymphotoxin-β pathway can be reproduced in several cellular models, including the full length replicon and HCV-infected primary human hepatocytes. We have identified NS5B, the HCV RNA dependent RNA polymerase, as the viral protein responsible for this phenotype and shown that pharmacological inhibition of its activity alleviates activation of the pro-inflammatory pathway. These results open new perspectives in understanding the inflammatory mechanisms linked to HCV infection and tumorigenesis.

Human mesenchymal stem cells (MSCs) have been used in cell-based therapy to promote revascularization after peripheral or myocardial ischemia. High levels of reactive oxygen species (ROS) are involved in the senescence and apoptosis of MSCs, causing defective neovascularization. Here, we examined the effect of the natural antioxidant lycopene on oxidative stress-induced apoptosis in MSCs. Although H2O2 (200 μM) increased intracellular ROS levels in human MSCs, lycopene (10 μM) pretreatment suppressed H2O2-induced ROS generation and increased survival. H2O2-induced ROS increased the levels of phosphorylated p38 mitogen activated protein kinase (MAPK), Jun-N-terminal kinase (JNK), ataxia telangiectasia mutated (ATM), and p53, which were inhibited by lycopene pretreatment. Furthermore, lycopene pretreatment decreased the expression of cleaved poly (ADP ribose) polymerase-1 (PARP-1) and caspase-3 and increased the expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), which were induced by H2O2 treatment. Moreover, lycopene significantly increased manganese superoxide dismutase (MnSOD) expression and decreased cellular ROS levels via the PI3K-Akt pathway. Our findings show that lycopene pretreatment prevents ischemic injury by suppressing apoptosis-associated signal pathway and enhancing anti-oxidant protein, suggesting that lycopene could be developed as a beneficial broad-spectrum agent for the successful MSC transplantation in ischemic diseases.

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health.

Pancreatic acinar cells possess very high protein synthetic rates as they need to produce and secrete large amounts of digestive enzymes. Acinar cell damage and dysfunction cause malnutrition and pancreatitis, and inflammation of the exocrine pancreas that promotes development of pancreatic ductal adenocarcinoma (PDAC), a deadly pancreatic neoplasm. The cellular and molecular mechanisms that maintain acinar cell function and whose dysregulation can lead to tissue damage and chronic pancreatitis are poorly understood. It was suggested that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis, but it is unknown whether impaired autophagy is a cause or a consequence of pancreatitis. To address this question, we generated Atg7(Δpan) mice that lack the essential autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. Atg7(Δpan) mice exhibit severe acinar cell degeneration, leading to pancreatic inflammation and extensive fibrosis. Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum (ER) stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in protein synthetic capacity that is accompanied by loss of rough ER. Atg7(Δpan) mice also exhibit spontaneous activation of regenerative mechanisms that initiate acinar-to-ductal metaplasia (ADM), a process that replaces damaged acinar cells with duct-like structures.

Foreign body (FB) ingestion is common in children, and button battery (BB) ingestion has been increasing in recent years. This study was to identify factors related to outcomes of FB ingestion, particularly BBs in the stomach. We evaluated whether the current recommendations are appropriate and aimed to suggest indications for endoscopic removal of BB in the stomach in young children.

IkappaB kinase beta (IKKbeta), required for NF-kappaB activation, links chronic inflammation with carcinogenesis. We investigated whether IKKbeta is involved in chemically induced liver cancer, a model not involving overt inflammation. Surprisingly, mice lacking IKKbeta only in hepatocytes (Ikkbeta(Deltahep) mice) exhibited a marked increase in hepatocarcinogenesis caused by diethylnitrosamine (DEN). This correlated with enhanced reactive oxygen species (ROS) production, increased JNK activation, and hepatocyte death, giving rise to augmented compensatory proliferation of surviving hepatocytes. Brief oral administration of an antioxidant around the time of DEN exposure blocked prolonged JNK activation and compensatory proliferation and prevented excessive DEN-induced carcinogenesis in Ikkbeta(Deltahep) mice. Decreased hepatocarcinogenesis was also found in mice lacking IKKbeta in both hepatocytes and hematopoietic-derived Kupffer cells. These mice exhibited reduced hepatocyte regeneration and diminished induction of hepatomitogens, which were unaltered in Ikkbeta(Deltahep) mice. IKKbeta, therefore, orchestrates inflammatory crosstalk between hepatocytes and hematopoietic-derived cells that promotes chemical hepatocarcinogenesis.

Human mesenchymal stem cells (hMSCs) are a potent source of cell-based regenerative therapeutics used to treat patients with ischemic disease. However, disease-induced oxidative stress disrupts mitochondrial homeostasis in transplanted hMSCs, resulting in hMSC apoptosis and reducing their efficacy post-transplantation. To address this issue, we evaluated the effects of melatonin on cellular defense mechanisms and mitophagy in hMSCs subjected to oxidative stress. H2O2-induced oxidative stress increases the levels of reactive oxygen species and reduces membrane potential in hMSCs, leading to mitochondrial dysfunction and cell death. Oxidative stress also decreases the expression of 70-kDa heat shock protein 1L (HSPA1L), a molecular chaperone that assists in the recruitment of parkin to the autophagosomal mitochondrial membrane. Decreased expression of HSPA1L destabilizes parkin, thereby impairing mitophagy. Our results indicate that treating hMSCs with melatonin significantly inhibited mitochondrial dysfunction induced by oxidative stress, which decreased hMSCs apoptosis. In damaged hMSCs, treatment with melatonin increased the levels of HSPA1L, which bound to parkin. The interaction between HSPA1L and parkin increased membrane potential and levels of oxidative phosphorylation, resulting in enhanced mitophagy. Our results indicate that melatonin increased the expression of HSPA1L, thereby upregulating mitophagy and prolonging cell survival under conditions of oxidative stress. In this study, we have shown that melatonin, a readily available compound, can be used to improve hMSC-based therapies for patients with pathologic conditions involving oxidative stress.

Analysis of human pathology led Braak to postulate that α-synuclein (α-syn) pathology could spread from the gut to brain via the vagus nerve. Here, we test this postulate by assessing α-synucleinopathy in the brain in a novel gut-to-brain α-syn transmission mouse model, where pathological α-syn preformed fibrils were injected into the duodenal and pyloric muscularis layer. Spread of pathologic α-syn in brain, as assessed by phosphorylation of serine 129 of α-syn, was observed first in the dorsal motor nucleus, then in caudal portions of the hindbrain, including the locus coeruleus, and much later in basolateral amygdala, dorsal raphe nucleus, and the substantia nigra pars compacta. Moreover, loss of dopaminergic neurons and motor and non-motor symptoms were observed in a similar temporal manner. Truncal vagotomy and α-syn deficiency prevented the gut-to-brain spread of α-synucleinopathy and associated neurodegeneration and behavioral deficits. This study supports the Braak hypothesis in the etiology of idiopathic Parkinson's disease (PD).

Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors.

Tumor necrosis factor (TNF) is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, because TNF does not kill normal IECs. Here, we investigated how chronic nuclear factor (NF)- κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice.