Death receptor 5 internalization is required for lysosomal permeabilization by TRAIL in malignant liver cell lines.
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.
Akazawa
Yuko
Y
Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
Mott
Justin L
JL
Bronk
Steven F
SF
Werneburg
Nathan W
NW
Kahraman
Alisan
A
Guicciardi
Maria Eugenia
ME
Meng
Xue Wei
XW
Kohno
Shigeru
S
Shah
Vijay H
VH
Kaufmann
Scott H
SH
McNiven
Mark A
MA
Gores
Gregory J
GJ
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
D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C105190:TNFRSF10A protein, human