ABSTRACT
Autophagy is responsible for the bulk degradation of cytosolic constituents and plays an essential role in the intestinal epithelium by controlling beneficial host-bacterial relationships. Atg5 and Atg7 are thought to be critical for autophagy. However, Atg5- or Atg7-deficient cells still form autophagosomes and autolysosomes, and are capable of removing proteins or bacteria. Here, we report that human TRIM31 (tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting lipopolysaccharide-induced Atg5/Atg7-independent autophagy. TRIM31 directly interacts with phosphatidylethanolamine in a palmitoylation-dependent manner, leading to induction of autolysosome formation. Depletion of endogenous TRIM31 significantly increases the number of intestinal epithelial cells containing invasive bacteria. Crohn's disease patients display TRIM31 downregulation. Human cytomegalovirus-infected intestinal cells show a decrease in TRIM31 expression as well as a significant increase in bacterial load, reversible by the introduction of wild-type TRIM31. We provide insight into an alternative autophagy pathway that protects against intestinal pathogenic bacterial infection.
Subject(s)
Autophagy/physiology , Crohn Disease/pathology , Epithelial Cells/metabolism , Intestinal Mucosa/physiology , Tripartite Motif Proteins/physiology , Ubiquitin-Protein Ligases/physiology , Adolescent , Adult , Autophagy/drug effects , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Autophagy-Related Protein 7/genetics , Autophagy-Related Protein 7/metabolism , Bacterial Load , Colon/microbiology , Colon/pathology , Crohn Disease/microbiology , Cytomegalovirus , Down-Regulation , Epithelial Cells/microbiology , Female , Gene Knockout Techniques , Humans , Ileum/microbiology , Ileum/pathology , Intestinal Mucosa/cytology , Intestinal Mucosa/microbiology , Lipopolysaccharides/pharmacology , Lysosomes/metabolism , Lysosomes/microbiology , Male , Middle Aged , Mitochondria/metabolism , Phosphatidylethanolamines/metabolism , RNA, Small Interfering/metabolism , Shigella flexneri , Young AdultABSTRACT
Reactive oxygen species (ROS) play an important role in cell signaling pathway. Previously, we found that silica induced immediate ROS generation and sequential cellular responses such as kinase activation in Rat2 cells as well as an increase of intracellular calcium concentration in A549 cells. However, the detailed mechanism underlying the immediate ROS generation induced by silica in fibroblast cells remains to be elucidated. Therefore, in the present study, we investigated the mechanism of ROS generation by silica within Rat2 fibroblast cells by examining the effects of a diverse group of inhibitors for the enzymes related with signal transduction events. Inhibitors for protein tyrosine kinase (PTK), phospholipase C (PLC), protein kinase C (PKC) and calmodulin (CaM) kinase II effectively suppressed ROS generation in silica-stimulated Rat2 cells, whereas those for protein kinase A and phospholipase A(2) did not. Diphenyleneiodonium chloride (DPI), an inhibitor for NADPH oxidase was also found to be effective in inhibiting silica-induced ROS generation. These results suggest that PTK, PLC, PKC, CaM kinase II, and NADPH oxidase are all involved in signal transduction pathways for ROS generation in silica-stimulated Rat2 cells.
