The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.
Olga A. Mareninova, Kip Hermann, Samuel W. French, Mark S. O’Konski, Stephen J. Pandol, Paul Webster, Ann H. Erickson, Nobuhiko Katunuma, Fred S. Gorelick, Ilya Gukovsky, Anna S. Gukovskaya
Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1–/– and Taldo1+/– mice spontaneously developed HCC, and Taldo1–/– mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1–/– livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced β-catenin phosphorylation and enhanced c-Jun expression in Taldo1–/– livers reflected adaptation to oxidative stress. Taldo1–/– hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1–/– mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.
Robert Hanczko, David R. Fernandez, Edward Doherty, Yueming Qian, Gyorgy Vas, Brian Niland, Tiffany Telarico, Adinoyi Garba, Sanjay Banerjee, Frank A. Middleton, Donna Barrett, Maureen Barcza, Katalin Banki, Steve K. Landas, Andras Perl
The accumulation of certain species of bacteria in the intestine is involved in both tissue homeostasis and immune-mediated pathologies. The host mechanisms involved in controlling intestinal colonization with commensal bacteria are poorly understood. We observed that under specific pathogen–free or germ-free conditions, intragastric administration of Pseudomonas aeruginosa, E. coli, Staphylococcus aureus, or Lactobacillus gasseri resulted in increased colonization of the small intestine and bacterial translocation in mice lacking Cd1d, an MHC class I–like molecule, compared with WT mice. In contrast, activation of Cd1d-restricted T cells (NKT cells) with α-galactosylceramide caused diminished intestinal colonization with the same bacterial strains. We also found prominent differences in the composition of intestinal microbiota, including increased adherent bacteria, in Cd1d–/– mice in comparison to WT mice under specific pathogen–free conditions. Germ-free Cd1d–/– mice exhibited a defect in Paneth cell granule ultrastructure and ability to degranulate after bacterial colonization. In vitro, NKT cells were shown to induce the release of lysozyme from intestinal crypts. Together, these data support a role for Cd1d in regulating intestinal colonization through mechanisms that include the control of Paneth cell function.
Edward E.S. Nieuwenhuis, Tetsuya Matsumoto, Dicky Lindenbergh, Rob Willemsen, Arthur Kaser, Ytje Simons-Oosterhuis, Sylvia Brugman, Keizo Yamaguchi, Hiroki Ishikawa, Yuji Aiba, Yasuhiro Koga, Janneke N. Samsom, Kenshiro Oshima, Mami Kikuchi, Johanna C. Escher, Masahira Hattori, Andrew B. Onderdonk, Richard S. Blumberg
HIF transcription factors (HIF-1 and HIF-2) are central mediators of cellular adaptation to hypoxia. Because the resting partial pressure of oxygen is low in the intestinal lumen, epithelial cells are believed to be mildly hypoxic. Having recently established a link between HIF and the iron-regulatory hormone hepcidin, we hypothesized that HIFs, stabilized in the hypoxic intestinal epithelium, may also play critical roles in regulating intestinal iron absorption. To explore this idea, we first established that the mouse duodenum, the site of iron absorption in the intestine, is hypoxic and generated conditional knockout mice that lacked either Hif1a or Hif2a specifically in the intestinal epithelium. Using these mice, we found that HIF-1α was not necessary for iron absorption, whereas HIF-2α played a crucial role in maintaining iron balance in the organism by directly regulating the transcription of the gene encoding divalent metal transporter 1 (DMT1), the principal intestinal iron transporter. Specific deletion of Hif2a led to a decrease in serum and liver iron levels and a marked decrease in liver hepcidin expression, indicating the involvement of an induced systemic response to counteract the iron deficiency. This finding may provide a basis for the development of new strategies, specifically in targeting HIF-2α, to improve iron homeostasis in patients with iron disorders.
Maria Mastrogiannaki, Pavle Matak, Brian Keith, M. Celeste Simon, Sophie Vaulont, Carole Peyssonnaux
Zhongsheng Peng, Pier Andrea Borea, Katia Varani, Tuere Wilder, Herman Yee, Luis Chiriboga, Michael R. Blackburn, Gianfranco Azzena, Giuseppe Resta, Bruce N. Cronstein
Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5′-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5′-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.
Zhongsheng Peng, Pier Andrea Borea, Tuere Wilder, Herman Yee, Luis Chiriboga, Michael R. Blackburn, Gianfranco Azzena, Giuseppe Resta, Bruce N. Cronstein
The epithelial anion channel CFTR interacts with multiple PDZ domain–containing proteins. Heterologous expression studies have demonstrated that the Na+/H+ exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3– secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3– secretory rates and blocked β2-adrenergic receptor (β2-AR) stimulation. Conversely, Nherf2–/– mice displayed augmented FSK-stimulated HCO3– secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3– secretion in WT mice, this effect was lost in Nherf2–/– mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3– secretion. In addition, laser microdissection and quantitative PCR revealed that the β2-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the β2-AR and CFTR was reduced in the Nherf1–/– mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3– secretion: while NHERF1 is an obligatory linker for β2-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.
Anurag Kumar Singh, Brigitte Riederer, Anja Krabbenhöft, Brigitte Rausch, Janina Bonhagen, Ulrich Lehmann, Hugo R. de Jonge, Mark Donowitz, Chris Yun, Edward J. Weinman, Olivier Kocher, Boris M. Hogema, Ursula Seidler
The proinflammatory cytokine IL-6 seems to have an important role in the intestinal inflammation that characterizes inflammatory bowel diseases (IBDs) such as Crohn disease and ulcerative colitis. However, little is known about the molecular mechanisms regulating IL-6 production in IBD. Here, we assessed the role of the transcriptional regulator IFN regulatory factor–4 (IRF4) in this process. Patients with either Crohn disease or ulcerative colitis exhibited increased IRF4 expression in lamina propria CD3+ T cells as compared with control patients. Consistent with IRF4 having a regulatory function in T cells, in a mouse model of IBD whereby colitis is induced in RAG-deficient mice by transplantation with CD4+CD45RBhi T cells, adoptive transfer of wild-type but not IRF4-deficient T cells resulted in severe colitis. Furthermore, IRF4-deficient mice were protected from T cell–dependent chronic intestinal inflammation in trinitrobenzene sulfonic acid– and oxazolone-induced colitis. In addition, IRF4-deficient mice with induced colitis had reduced mucosal IL-6 production, and IRF4 was required for IL-6 production by mucosal CD90+ T cells, which it protected from apoptosis. Finally, the protective effect of IRF4 deficiency could be abrogated by systemic administration of either recombinant IL-6 or a combination of soluble IL-6 receptor (sIL-6R) plus IL-6 (hyper–IL-6). Taken together, our data identify IRF4 as a key regulator of mucosal IL-6 production in T cell–dependent experimental colitis and suggest that IRF4 might provide a therapeutic target for IBDs.
Jonas Mudter, Lioubov Amoussina, Mirjam Schenk, Jingling Yu, Anne Brüstle, Benno Weigmann, Raja Atreya, Stefan Wirtz, Christoph Becker, Arthur Hoffman, Imke Atreya, Stefan Biesterfeld, Peter R. Galle, Hans A. Lehr, Stefan Rose-John, Christoph Mueller, Michael Lohoff, Markus F. Neurath
Chronic Helicobacter pylori infection is recognized as a cause of gastric cancer. H. pylori adhesion to gastric cells is mediated by bacterial adhesins such as sialic acid–binding adhesin (SabA), which binds the carbohydrate structure sialyl–Lewis x. Sialyl–Lewis x expression in the gastric epithelium is induced during persistent H. pylori infection, suggesting that H. pylori modulates host cell glycosylation patterns for enhanced adhesion. Here, we evaluate changes in the glycosylation-related gene expression profile of a human gastric carcinoma cell line following H. pylori infection. We observed that H. pylori significantly altered expression of 168 of the 1,031 human genes tested by microarray, and the extent of these alterations was associated with the pathogenicity of the H. pylori strain. A highly pathogenic strain altered expression of several genes involved in glycan biosynthesis, in particular that encoding β3 GlcNAc T5 (β3GnT5), a GlcNAc transferase essential for the biosynthesis of Lewis antigens. β3GnT5 induction was specific to infection with highly pathogenic strains of H. pylori carrying a cluster of genes known as the cag pathogenicity island, and was dependent on CagA and CagE. Further, β3GnT5 overexpression in human gastric carcinoma cell lines led to increased sialyl–Lewis x expression and H. pylori adhesion. This study identifies what we believe to be a novel mechanism by which H. pylori modulates the biosynthesis of the SabA ligand in gastric cells, thereby strengthening the epithelial attachment necessary to achieve successful colonization.
Nuno T. Marcos, Ana Magalhães, Bibiana Ferreira, Maria J. Oliveira, Ana S. Carvalho, Nuno Mendes, Tim Gilmartin, Steven R. Head, Céu Figueiredo, Leonor David, Filipe Santos-Silva, Celso A. Reis
Mutations in the RET gene are the primary cause of Hirschsprung disease (HSCR), or congenital intestinal aganglionosis. However, how RET malfunction leads to HSCR is not known. It has recently been shown that the binding of glial cell line–derived neurotrophic factor (GDNF) to GDNF family receptor α1 (GFRα1) activates RET and is essential for the survival of enteric neurons. In this study, we investigated Ret regulation of enteric neuron survival and its potential involvement in HSCR. Conditional ablation of Ret in postmigratory enteric neurons caused widespread neuronal death in the colon, which led to colonic aganglionosis. To further examine this finding, we generated a mouse model for HSCR by reducing Ret expression levels. These mice recapitulated the genetic and phenotypic features of HSCR and developed colonic aganglionosis due to impaired migration and successive death of enteric neural crest–derived cells. Death of enteric neurons was also induced in the colon, where reduction of Ret expression was induced after the period of enteric neural crest cell migration, indicating that diminished Ret expression directly affected the survival of colonic neurons. Thus, enteric neuron survival is sensitive to RET dosage, and cell death is potentially involved in the etiology of HSCR.
Toshihiro Uesaka, Mayumi Nagashimada, Shigenobu Yonemura, Hideki Enomoto