N-glycosylation of mouse TRAIL-R restrains TRAIL-induced apoptosis

Abstract

The sensitivity of cells to death receptor-induced apoptosis is commonly controlled by multiple checkpoints in order to limit induction of excessive or unnecessary death. Although cytotoxic in various cancer cells, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) does not trigger apoptosis in most non-transformed cells. The molecular nature of the checkpoints that normally protect the cells from TRAIL-induced death are not fully understood. Endoplasmic reticulum (ER) stress has been reported to switch the sensitivity of human cells to the cytotoxic effect of TRAIL, suggesting that this cellular state perturbs some of these protective mechanisms. We found that tunicamycin (TU), but no other ER stress inducers, sensitized mouse fibroblasts and hippocampal neuronal cells to TRAIL-induced apoptosis. Importantly, the sensitization was specific to TRAIL and not caused by differences in ER stress induction. Instead, it relied on the inhibition of N-glycosylation of the mouse TRAIL receptor (mTRAIL-R). Inhibition of N-glycosylation did not alter cell surface expression of mTRAIL-R but enhanced its ability to bind TRAIL, and facilitated mTRAIL-R oligomerization, which resulted in enhanced death-inducing signaling complex (DISC) formation and caspase-8 activation. Remarkably, reconstitution of mTRAIL-R-deficient cells with a version of mTRAIL-R mutated for the three N-glycosylation sites identified in its ectodomain confirmed higher sensitivity to TRAIL-induced apoptosis. Together, our results demonstrate that inhibition of N-glycosylation of mTRAIL-R, and not ER stress induction, sensitizes mouse cells to TRAIL-induced apoptosis. We therefore reveal a new mechanism restraining TRAIL cytotoxicity in mouse cells.

Fig. 1. Tunicamycin specifically sensitizes mouse cells to TRAIL-induced apoptosis.

a Cell death profiles of MEFs treated with 20 ng/ml recombinant mouse SuperKillerTRAIL (mTRAIL SK), 1 μg/ml tunicamycin (TU), or the combination of both. The percentage of cell death was measured over time using a Fluostar Omega fluorescence plate reader analyzing the SYTOX Green-positive cells. Error bars represent S.E.M. of three independent experiments. *P < 0.05; **P < 0.01. b MEFs were stimulated with TU in presence or absence of 20 ng/ml mTRAIL-SK for 15 h, and cell lysates were immunoblotted as indicated. Caspase cleavage products are indicated by arrowheads. Representative images of two independent experiments. c Cell death profile of MEFs pretreated with 20 μM Z-VAD-FMK (Z-VAD) or control (CTRL) followed by treatment with either mTRAIL-SK (20 ng/ml), TU, or TU + mTRAIL-SK for 18 h. Cell death was measured using a Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of two independent experiments. d Cell death profiles of MEFs stimulated with TU in combination or not with increasing doses (6, 20, and 200 ng/ml) of mTRAIL SK or mTRAIL for 20 h. Cell death was measured using a Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of two independent experiments. e Cell death profiles of MEF cells stimulated with 20 ng/ml mTRAIL-SK in combination or not with increasing doses of TU for 20 h. Cell death was measured using a Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of two independent experiments. f, g Cell death profiles of MEFs exposed to f 1 μM thapsigargin (THAP) or THAP + 20 ng/ml mTRAIL-SK; or g 0.5 μM brefeldin A (BFA) or BFA + 20 ng/ml mTRAIL-SK for 16 h and 20 h. Cell death was measured using a Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of three independent experiments. h MEFs were incubated with TU, THAP, or BFA for 4 and 6 h, and cell lysates were immunoblotted as indicated. Representative images of two independent experiments. i Cell death profiles of MEFs exposed to TU or 1.5 μg/ml translation inhibitor cycloheximide (CHX) in combination or not with 20 ng/ml of human (h) or mouse (m) TNF for 20 h. Cell death was measured using a Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of two independent experiments. j Cell death profiles of MEFs exposed to TU or 1.5 μg/ml CHX in combination or not with 20 ng/ml mTRAIL-SK or hFas Ligand (hFasL) for 16 h. Cell death was measured using a Fluostar Omega fluorescence plate reader. Results are representative of two independent experiments. Error bars represent S.E.M. of triplicates from a representative experiment. k Cell death profiles of HT22 cells treated for 24 h with 20 ng/ml mTRAIL-SK, or with TU or THAP in combination or not with mTRAIL-SK. Cell death was measured using a Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of two independent experiments. l Cell death profiles of 3T3 cells treated with mTRAIL-SK or with TU or THAP, in combination or not with mTRAIL-SK. Cell death was measured using an Incucyte ZOOM® system. Results are representative of two independent experiments. Error bars represent S.E.M. of duplicates from a representative experiment

Fig. 2. Tunicamycin induces appearance of a non-N-glycosylated form of mTRAIL-R.

a MEFs were treated with CTRL, TU, or THAP for 17 h, and cell lysates were immunoblotted for mTRAIL-R and β-tubulin. Asterisk indicates non-specific signal. Representative images of three independent experiments. b MEFs were transfected with non-silencing (NS) or mTRAIL-R specific siRNA, and then treated with TU for 17 h. Cell lysates were immunblotted for mTRAIL-R and β-tubulin. Asterisk indicates non-specific signal. Representative images of three independent experiments. c Cell lysates of CTRL- or TU-treated MEFs were incubated at 37 °C for 1 h in presence or absence of PNGase F (PNG), and then immunoblotted for mTRAIL-R and β-tubulin. Asterisk indicates non-specific signal. Representative images of two independent experiments. d MEF cells were treated with TU for increasing times and cell lysates were immunblotted for mTRAIL-R and β-tubulin. Asterisk indicates non-specific signal. Representative images of three independent experiments

Fig. 3. Non-N-glycosylated mTRAIL-R is expressed at the plasma membrane where it exhibits higher binding capacity for TRAIL.

a Cell surface expression of mTRAIL-R in MEFs treated for 7 h with CTRL or TU, and analyzed by flow cytometry using a FACSVerse cytometer. Isotype-PE (filled histogram), mTRAIL-PE (open histogram). Representative histogram of three independent experiments. b Immunoblots under non-reducing conditions of mTRAIL-R of a His-Tag pulldown on MEFs pre-treated with TU or control for 7 h followed by mTRAIL-SK (500 ng/ml) treatment for increasing times. Before loading, pulldown samples were treated (+PNG) or not with PNGase F in non-reducing conditions. Representative images of at least two independent experiments

Fig. 4. Non-N-glycosylated mTRAIL-R triggers ligand-dependent apoptosis in tunicamycin-treated cells.

a MEFs were treated for 6 h with control or TU followed by stimulation with mTRAIL-SK (20 ng/ml) for the indicated times. Cell lysates were then immunoblotted as indicated. Caspase-8 and c-FLIP cleavage products are indicated by arrowheads. Asterisk indicates non-specific signal. Representative images of three independent experiments. b MEFs were exposed to TU or control for 7 h followed by Biotin-ILZ-hTRAIL (biot-ILZ-hTRAIL) (500 ng/ml) or CTRL treatment for 15 min. Cell lysates were subjected to streptavidin (Strep) pulldown and immunoblotted for mTRAIL-R and FADD under non-reducing conditions. Left panels: Strep pulldown. Right panels: Input. HMW high molecular weight. Representative images of two independent experiments. c MEFs were exposed to TU or control for 7 h followed by mTRAIL-SK (20 ng/ml) or CTRL treatment for 2 h. Cell lysates were subjected to FADD immunoprecipitation (IP:FADD) and immunoblotted as indicated. mTRAIL-R was detected under non-reducing conditions. Left panels: IP:FADD. Right panels: Input. Caspase-8 cleavage products are indicated by arrowheads. HMW high molecular weight. Asterisk indicates non-specific signal. Representative images of two independent experiments

Fig. 5. mTRAIL-R is N-glycosylated on three asparagine residues in its ectodomain.

a Schematic presentation of the three putative N-glycosylation sites (NetGlyc 1.0 prediction) in the extracellular domain of mTRAIL-R. b Trail-R^−/− MEFs were transiently transfected with an empty vector (EV), wild-type (WT) mTRAIL-R, or N-glyc mTRAIL-R mutants pcDNA3 plasmids for 24 h, and cell lysates were immunoblotted for mTRAIL-R. Representative images of three independent experiments. c Trail-R^−/− MEFs were transiently transfected with an EV, WT mTRAIL-R, or N-glyc mTRAIL-R mutants pcDNA3 plasmids for 24 h in absence (CTRL) or presence of TU, and cell lysates were immunoblotted for mTRAIL-R. Representative images of three independent experiments

Fig. 6. N-glycosylation of mTRAIL-R restrains apoptosis induction.

a Trail-R^−/− MEFs were transiently transfected with 300 ng of pcDNA3 plasmids coding for a wild-type (WT) mTRAIL-R, or non-N-glyc mTRAIL-R N99/122/150Q mutant. After 24 h, the percentage of double positive mTRAIL-R+/Annexin V+ cells was analyzed by flow cytometry using a FACSVerse cytometer. Error bars represent S.E.M. of four independent experiments. *P < 0.05 in a ratio paired t test. b Trail-R^−/− MEFs were transiently transfected with 300 or 600 ng of pcDNA3 plasmids coding for WT or N99/122/150Q mutant mTRAIL-R. After 24 h, cell lysates were immunoblotted for mTRAIL-R in reducing vs non-reducing conditions. HMW high molecular weight. Representative images of at least two independent experiments. c Trail-R^−/− MEFs were stably transduced with viral particles coding for an inducible wild-type (iWT) mTRAIL-R, or non-N-glyc mTRAIL-R N99/122/150Q mutant (iN99/122/150Q). The cells were treated with doxycycline (Dox) at the indicated concentrations for 24 h, and cell lysates were immunoblotted for mTRAIL-R in non-reducing conditions and for actin in reducing conditions. Representative images of three independent experiments. d, e Cell death (d) and caspase-3 activity (e) profiles of Trail-R^−/− MEFs transduced as in c, and then treated with mTRAIL-SK (20 ng/ml), cycloheximide (CHX; 0.250 μg/ml), or the CHX/mTRAIL-SK combination for 24 h. Cell death and caspase-3 activity were measured using a Fluostar Omega fluorescence plate reader. Error bars represent S.E.M. of three (d) and two (e) independent experiments. **P < 0.01