Death Receptor 5 Internalization Is Required for Lysosomal Permeabilization by TRAIL in Malignant Liver Cell Lines

Yuko Akazawa, Justin L. Mott, Steven F. Bronk, Nathan W. Werneburg, Alisan Kahraman, Maria Eugenia Guicciardi, Xue Wei Meng, Shigeru Kohno, Vijay H. Shah, Scott H. Kaufmann, Mark A. McNiven, Gregory J. Gores

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Abstract

Background & Aims: 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.

Original languageEnglish (US)
Pages (from-to)2365-2376.e7
JournalGastroenterology
Volume136
Issue number7
DOIs
StatePublished - Jun 2009

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TNF-Related Apoptosis-Inducing Ligand Receptors
Endocytosis
Lysosomes
Apoptosis
Cell Line
Liver
Dynamins
Ligands
Small Interfering RNA
Clathrin
Cholangiocarcinoma
RNA Interference
Confocal Microscopy
Cytosol
Microscopy
Hepatocellular Carcinoma
Peptide Hydrolases
Tumor Necrosis Factor-alpha

ASJC Scopus subject areas

  • Hepatology
  • Gastroenterology

Cite this

Akazawa, Y., Mott, J. L., Bronk, S. F., Werneburg, N. W., Kahraman, A., Guicciardi, M. E., ... Gores, G. J. (2009). Death Receptor 5 Internalization Is Required for Lysosomal Permeabilization by TRAIL in Malignant Liver Cell Lines. Gastroenterology, 136(7), 2365-2376.e7. https://doi.org/10.1053/j.gastro.2009.02.071

Death Receptor 5 Internalization Is Required for Lysosomal Permeabilization by TRAIL in Malignant Liver Cell Lines. / Akazawa, Yuko; Mott, Justin L.; Bronk, Steven F.; Werneburg, Nathan W.; Kahraman, Alisan; Guicciardi, Maria Eugenia; Meng, Xue Wei; Kohno, Shigeru; Shah, Vijay H.; Kaufmann, Scott H.; McNiven, Mark A.; Gores, Gregory J.

In: Gastroenterology, Vol. 136, No. 7, 06.2009, p. 2365-2376.e7.

Research output: Contribution to journalArticle

Akazawa, Y, Mott, JL, Bronk, SF, Werneburg, NW, Kahraman, A, Guicciardi, ME, Meng, XW, Kohno, S, Shah, VH, Kaufmann, SH, McNiven, MA & Gores, GJ 2009, 'Death Receptor 5 Internalization Is Required for Lysosomal Permeabilization by TRAIL in Malignant Liver Cell Lines', Gastroenterology, vol. 136, no. 7, pp. 2365-2376.e7. https://doi.org/10.1053/j.gastro.2009.02.071
Akazawa, Yuko ; Mott, Justin L. ; Bronk, Steven F. ; Werneburg, Nathan W. ; Kahraman, Alisan ; Guicciardi, Maria Eugenia ; Meng, Xue Wei ; Kohno, Shigeru ; Shah, Vijay H. ; Kaufmann, Scott H. ; McNiven, Mark A. ; Gores, Gregory J. / Death Receptor 5 Internalization Is Required for Lysosomal Permeabilization by TRAIL in Malignant Liver Cell Lines. In: Gastroenterology. 2009 ; Vol. 136, No. 7. pp. 2365-2376.e7.
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abstract = "Background & Aims: 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.",
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T1 - Death Receptor 5 Internalization Is Required for Lysosomal Permeabilization by TRAIL in Malignant Liver Cell Lines

AU - Akazawa, Yuko

AU - Mott, Justin L.

AU - Bronk, Steven F.

AU - Werneburg, Nathan W.

AU - Kahraman, Alisan

AU - Guicciardi, Maria Eugenia

AU - Meng, Xue Wei

AU - Kohno, Shigeru

AU - Shah, Vijay H.

AU - Kaufmann, Scott H.

AU - McNiven, Mark A.

AU - Gores, Gregory J.

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N2 - Background & Aims: 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.

AB - Background & Aims: 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.

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