Volume 136, Issue 7, Pages 2365-2376.e7 (June 2009) 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  Gastroenterology  Volume 136, Issue 7, Pages 2365-2376.e7 (June 2009) DOI: 10.1053/j.gastro.2009.02.071 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 DR5 contributes more than DR4 to TRAIL-mediated lysosomal permeabilization and apoptosis. (A) Huh-7 cells stably transfected with shRNA complementary to DR4 or DR5, or untransfected Huh-7 (wild-type), were treated with 4 ng/mL TRAIL for 8 hours. Apoptosis was assessed by morphologic criteria after DAPI staining. Error bars depict ± SEM from 3 or more independent experiments. *P < .05, TRAIL-treated DR5-shRNA vs wild type. (Insert) Immunoblotting showing effective knockdown of DR4 and DR5 on total cell lysates. (B) After cells were treated with TRAIL for 8 hours, activity of activity of effector caspases 3 and 7 was measured by a fluorogenic assay. Data are expressed as fold increase of relative fluorescence units (RFLU) over control value (untreated cells), which was arbitrarily set to 1, and represent the mean ± SE. *P < .05, TRAIL-treated DR5-shRNA vs wild type. (C) Huh-7 cells stably transfected with shRNA targeting DR4 or DR5, or untransfected Huh-7 cells (wild-type [WT]), were treated with 4 ng/mL TRAIL for 4 hours. Localization of cathepsin B was visualized by immunofluorescence by using a confocal microscope (64×), and (D) cells were scored for punctate or diffuse appearance of the antigen. *P < .05, TRAIL-treated DR5-shRNA vs wild type. Experimental procedures relevant to this figure are explained in the supplementary methods (see supplemental material online at www.gastrojournal.org). Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 TRAIL undergoes cellular internalization upon ligand stimulation. (A) Huh-7 cells were incubated with FLAG-TRAIL cross-linked by anti-FLAG M2 antibody for 30 minutes on ice, followed by incubation at 37°C for the indicated time. Cells were then fixed, incubated with fluorescent secondary antibody, and analyzed by confocal microscopy. (B) To visualize only internalized TRAIL, Huh-7 cells stably transfected with shRNA complementary to DR4 or DR5, or untransfected Huh-7 (wild-type), were treated with FLAG-TRAIL and mouse monoclonal M2 antibody as illustrated in A and then washed with 0.2 mol/L acetic acid before fixation to remove the membrane-associated FLAG-TRAIL. Cells were then incubated with fluorescent anti-mouse immunoglobulin G. Blue fluorescence, DAPI staining; green fluorescence, internalized FLAG-TRAIL. (C) Quantification of internalized FLAG-TRAIL. Cells were treated as illustrated in B. Data were expressed as the area of the cell multiplied by the average fluorescence intensity in the cell above the background. *P < .01, TRAIL-treated DR5-shRNA vs TRAIL-treated wild type. (D) Cells were transiently transfected with a plasmid expressing EGFP-tagged DR5 (DR5-EGFP). Cells were observed on a heated microscope stage at 37°C with or without 20 ng/mL of TRAIL for 30 minutes. Disappearance of membrane DR5 was observed by TIRF microscopy. (E) The percentage receptor remaining on the cell surface was assessed by quantification of discrete fluorescent foci. *P < .01, TRAIL-treated DR5-EGFP vs DR4-EGFP. Error bars depict ± SEM from 3 or more independent experiments. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Mutation of predicted internalization motif in DR5 blocks TRAIL-mediated endocytosis of DR5. Huh-7 cells were transiently transfected with wild-type DR5-EGFP or mutant DR5-EGFP for 48 hours. Cells were then treated with diluent or TRAIL (20 ng/mL) for 30 minutes at 37°C on the heated stage of a TIRF microscope. The percentage of surface receptor remaining at 30 minutes imaged by TIRF was assessed by quantification of fluorescent particles. *P < .05, TRAIL-treated wild-type DR5-EGFP vs TRAIL-treated mutant DR5-EGFP. Error bars depict ± SEM from 3 or more independent experiments. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 (A) Huh-7 cells were transduced with either adenovirus encoding K44A Dyn or empty vector (Empty Ad). Twenty-four hours after infection, cells were treated with FLAG-TRAIL (400 ng/mL), fixed, stained with anti-FLAG antibody, and analyzed by confocal microscopy. (B) Twenty-four hours after transient transfection with DR5-EGFP, cells were transduced with either K44A Dyn or empty adenovirus for 24 hours. Cells were then treated with diluent or TRAIL (20 ng/mL) for 30 minutes at 37°C on the heated stage of a TIRF microscope. (C) The percentage of surface receptor remaining at 30 minutes imaged by TIRF was assessed by quantification of fluorescent particles. *P < .05, TRAIL-treated empty ad vs K44A Dyn ad. Error bars depict ± SEM from 3 or more independent experiments. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 K44A dynamin inhibits TRAIL-mediated cathepsin B release from lysosomes and cytotoxicity in Huh-7 cells. (A) Huh-7 cells infected with wild-type dynamin, K44A dynamin adenovirus, or empty adenovirus were incubated with TRAIL (4 ng/mL) for 8 hours. Apoptosis was assessed by morphologic criteria after DAPI staining. Control: not infected. *P < .05, TRAIL-treated K44A Ad. vs empty Ad. (B) Activation of effector caspases 3 and 7 was measured by a fluorogenic assay. *P < .05, TRAIL-treated K44A Ad vs empty Ad. (C) After cells were infected with adenovirus and treated with TRAIL as shown in A, annexin V study was performed using confocal microscopy. *P < .01, TRAIL-treated K44A Ad vs empty Ad. (D) Beginning 24 hours after transduction with K44A dynamin or empty adenovirus, Huh-7 cells were treated with TRAIL (4 ng/mL) for 4 hours. Cells were scored for punctate or diffuse appearance of cathepsin B as shown in Figure 2A. *P < .05, TRAIL-treated K44A Dyn Ad vs empty Ad. Experimental procedures relevant to this figure are explained in the supplementary methods (see supplemental material online at www.gastrojournal.org). Error bars depict ± SEM from 3 or more independent experiments. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 DR5 becomes localized to the lysosomes upon TRAIL treatment. (A) Huh-7 cells were transfected with DR5-EGFP for 48 hours and then treated with 20 ng/mL TRAIL for 1 hour. LysoTracker Red was added 30 minutes before cells were observed by confocal microscopy. N, nucleus. (B) Colocalization is calculated as a ratio of colocalized DR5-EGFP and LysoTracker Red compared with total EGFP. *P < .05, untreated Huh-7 cells vs TRAIL-treated Huh-7 cells. (C) After cells were treated with diluent or 20 ng/mL TRAIL for 1 hour, lysosomes were isolated. Collected lysosomes and whole cell lysates were subjected to immunoblot analysis for indicated proteins. The purity of the lysosomal preparation was verified by the absence of the ER (calnexin), early endosome (EEA-1), and mitochondrial (cytochrome c oxidase) markers. Experimental procedures relevant to this figure are explained in the supplementary methods (see supplemental material online at www.gastrojournal.org). Error bars depict ± SEM from 3 or more independent experiments. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Rab7 is required for lysosomal trafficking of DR5 and apoptosis mediated by TRAIL. (A) Huh-7 cells were transfected with Rab7 siRNA or scrambled siRNA for 48 hours. Cells were lysed and were subjected to immunoblot analysis for Rab7, cathepsin B, and cathepsin D. (B) Huh-7 cells were transfected with Rab7 siRNA. After 24 hours, cells were transfected with DR5-EGFP for another 24 hours. Cells were then treated with 4 ng/mL TRAIL for 60 minutes. LysoTracker Red was added 30 minutes before cells were observed by confocal microscopy. Colocalization was calculated as a ratio of colocalized DR5-EGFP and LysoTracker Red compared with total EGFP. *P < .05, TRAIL-treated Rab7 siRNA vs control. (C) Untransfected (control) and Huh-7 cells transfected with Rab7 siRNA or scrambled siRNA were treated with TRAIL for 8 hours. Apoptosis was assessed by nuclear morphologic changes using nuclear binding dye DAPI. *P < .05, TRAIL-treated Rab7 siRNA transfected cells vs scrambled siRNA transfected cells. (D) Untransfected (control) and Huh-7 cells transfected with Rab7 siRNA or scrambled siRNA were treated with TRAIL for 4 hours. Cellular compartmentation of cathepsin D was assessed by immunofluorescence and confocal microscopy. Cells were scored as punctate or diffuse based on cellular localization of the antigen. *P < .05, TRAIL-treated Rab7 siRNA transfected cells vs scrambled siRNA transfected cells. Experimental procedures relevant to this figure are explained in the supplementary methods (see supplemental material online at www.gastrojournal.org). Error bars depict ± SEM from 3 or more independent experiments. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Schematic representation of the proposed model for TRAIL-induced apoptosis through internalization of TRAIL ligand complex and lysosomal permeabilization. See text for details. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Monoclonal agonistic antibody against DR5 induces more apoptosis than monoclonal agonistic antibody against DR4. HuH-7 cells were treated with either monoclonal agonistic antibody against DR4 (DR4-mAb; 1 μg) or DR5 (DR5-mAb; 1 μg) for the indicated time before assessment of apoptosis. *P < .05, TRAIL-treated DR5-mAb vs DR4-mAb. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 Mutation of predicted internalization motif in DR5 does not alter cell membrane DR5 expression. Huh-7 cells were transiently transfected with wild-type (wt)-DR5-EGFP or mutant (mut)-DR5-EGFP for 48 hours. Cells were then observed on the heated stage of a TIRF microscope. DR5 membrane expression was assessed by quantification of fluorescent particles. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 Dominant negative dynamin (K44A Dyn) does not alter cell surface expression of DR5. Cells were transduced with either K44A dynamin adenovirus or empty adenovirus for 24 hours. Cells not subjected to adenovirus infection were also used as a control (Not infected). Cells were fixed, incubated with anti-DR5 followed by fluorescent secondary antibody, and analyzed by flow cytometry. Negative control: secondary antibody only. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 4 Dynamin-dependent internalization of TRAIL is necessary for TRAIL-induced apoptosis in HNU-499 cells, Mz-ChA-1 cells, and HuCCT-1 cells. (A and B) Beginning 24 hours after infection with K44A adenovirus or empty adenovirus, cells were incubated with TRAIL (8–15 ng/mL) for 8 hours. (A) Apoptosis was assessed by morphologic criteria after DAPI staining or (B) caspase-3/7 activity was measured by a fluorogenic assay. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 5 Dynamin-dependent internalization of TRAIL is not necessary for TRAIL-induced apoptosis in HeLa cells. (A and B) Beginning 24 hours after infection with K44A adenovirus or empty adenovirus, HeLa cells were incubated with TRAIL (4 ng/mL) for 8 hours. (A) Apoptosis was assessed by morphologic criteria after DAPI staining or (B) caspase-3/7 activity was measured by a fluorogenic assay. (C and D) HeLa and Huh-7 cells were treated with 4 ng/mL TRAIL for 4 hours. (C) Cathepsin D was visualized by immunofluorescence by using a confocal microscope, and (D) cells were scored for punctate or diffuse appearance of the antigen. *P < .05, TRAIL-treated Huh-7 cells vs HeLa cells. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 6 (A and B) Rab7 siRNA does not alter localization of cathepsin B to the lysosomes. Huh-7 cells were transfected with Rab7 siRNA. After 24 hours, cells were transfected with a plasmid expressing cathepsin B-EGFP for another 24 hours. LysoTracker Red was added 30 minutes before cells were observed by confocal microscopy. Colocalization was calculated as the ratio of colocalized cathepsin B-EGFP and LysoTracker Red compared with total EGFP. Gastroenterology 2009 136, 2365-2376.e7DOI: (10.1053/j.gastro.2009.02.071) Copyright © 2009 AGA Institute Terms and Conditions