title: Death receptor 5 internalization is required for lysosomal permeabilization by TRAIL in malignant liver cell lines.
abstract: OBJECTIVE
  Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity in hepatocellular carcinoma cells is mediated by lysosomal permeabilization. Our aims were to determine which TRAIL receptor, death receptor (DR) 4 or DR5, mediates lysosomal permeabilization and assess whether receptor endocytosis followed by trafficking to lysosomes contributes in this process.
  
  
  METHODS
  TRAIL ligand internalization in Huh-7 cells was examined by confocal microscopy using Flag-tagged TRAIL, whereas DR4- and DR5-enhanced green fluorescent protein internalization was assessed by total internal reflection microscopy. Clathrin-dependent endocytosis was inhibited by expressing dominant negative dynamin.
  
  
  RESULTS
  Although Huh-7 cells express both TRAIL receptors, short hairpin RNA silencing of DR5 but not DR4 attenuated TRAIL-mediated lysosomal permeabilization and apoptosis. The TRAIL/DR5 complex underwent rapid cellular internalization upon ligand stimulation, whereas the TRAIL/DR4 complex was not efficiently internalized. DR5-enhanced green fluorescent protein internalization was dependent on a dileucine-based internalization motif. Endocytosis of the TRAIL/DR5 complex was dynamin dependent and was required for rapid lysosomal permeabilization and apoptosis in multiple malignant hepatocellular and cholangiocarcinoma cell lines. Upon TRAIL treatment, DR5 colocalized with lysosomes after internalization. Inhibition of DR5 trafficking to lysosomes by Rab7 small interfering RNA also reduced TRAIL-mediated lysosomal disruption and apoptosis.
  
  
  CONCLUSIONS
  TRAIL-mediated endocytosis of DR5 with trafficking to lysosomes contributes to lysosomal protease release into the cytosol and efficient apoptosis in malignant liver cell lines.
authors.affiliation: Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Akazawa
authors.forename: Justin L
authors.initials: JL
authors.lastname: Mott
authors.forename: Steven F
authors.initials: SF
authors.lastname: Bronk
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Werneburg
authors.forename: Alisan
authors.initials: A
authors.lastname: Kahraman
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Guicciardi
authors.forename: Xue Wei
authors.initials: XW
authors.lastname: Meng
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kohno
authors.forename: Vijay H
authors.initials: VH
authors.lastname: Shah
authors.forename: Scott H
authors.initials: SH
authors.lastname: Kaufmann
authors.forename: Mark A
authors.initials: MA
authors.lastname: McNiven
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gores
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D045744:Cell Line, Tumor; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D011336:Probability; D034622:RNA Interference / Q000502:physiology; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C105190:TNFRSF10A protein, human