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. 2006 Apr;26(8):3071-84.
doi: 10.1128/MCB.26.8.3071-3084.2006.

Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

Ping Hu  1 , Zhang Han, Anthony D Couvillon, Randal J Kaufman, John H Exton
Affiliations

Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

Ping Hu et al. Mol Cell Biol. 2006 Apr.

Abstract

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.

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Figures

FIG. 1.
FIG. 1.
IRE1α is required for ER stress-induced NF-κB activation. (A) ER stress induces IκBα degradation. MCF-7, H1299, and PC-3 cells were exposed to thapsigargin (2 μM) or tunicamycin (2 μg/ml) for the indicated times, and cell extracts were subjected to SDS-PAGE for Western blotting with anti-IκBα and anti-β-actin antibodies. (B) ER stress induces IκBα phosphorylation. MCF-7 cells were treated with thapsigargin or tunicamycin for the indicated times, and phosphorylated IκBα was examined by Western blotting. (C) ER stress activates NF-κB DNA binding activity. MCF-7, H1299, and PC-3 cells were treated with thapsigargin (2 μM) or tunicamycin (2 μg/ml) for various times as indicated. These concentrations were used in all subsequent experiments. Nuclear extracts were prepared, and 5 μg of nuclear extract from each sample was used to analyze NF-κB DNA binding activity by EMSA with an NF-κB probe. Sp-1 DNA binding activity in each sample was detected as a loading control. (D) Impaired activation of NF-κB by ER stress in IRE1α knockdown cells. MCF-7 cells were transfected with a control siRNA and an siRNA pool specific to human IRE1α for 48 h. Cell lysates were then analyzed for IRE1α level by Western blotting as shown in the upper panel. The same treated MCF-7 cells were subjected to treatment with thapsigargin or tunicamycin for various times as indicated. Whole-cell extracts and nuclear extracts were prepared, and IκBα degradation and NF-κB DNA binding activity were examined by Western blotting and EMSA with an anti-IκBα antibody and an NF-κB probe, respectively. (E) TNF-α-induced NF-κB activation in IRE1α−/− and IRE1α+/+ MEFs. Mouse wild-type and IRE1α−/− fibroblasts were incubated with TNF-α (15 ng/ml) for the indicated times. The levels of IκBα and β-actin were examined by Western blotting. (F) Expression of IRE1α reconstitutes impaired ER stress-induced IκBα degradation and NF-κB activation in IRE1α−/− fibroblast cells. IRE1α−/− MEFs were transfected with a control and a T7-tagged IRE1α vector for 36 h, and cell lysates were analyzed for IRE1α-T7 level by Western blotting as shown in the upper panel. IRE1α+/+, IRE1α−/−, and IRE1α−/− (IRE1α) fibroblast cells were incubated with thapsigargin or tunicamycin as shown in the lower panel. NF-κB activity was measured by EMSA with a NF-κB probe. TG, thapsigargin; TU, tunicamycin; Conti, control siRNA.
FIG.2.
FIG.2.
ER stress induces formation of IRE1α and IKK complex. (A) Interaction of IRE1α with IKKβ in vivo. T7-tagged IRE1α and Flag-tagged IKKβ were cotransfected into HEK293 cells. Cell lysates were immunoprecipitated with anti-Flag antibody or T7 antibody, respectively, and then subjected to SDS-PAGE for Western blotting with anti-T7 antibody or anti-Flag antibody. Five percent of the cell extract from each sample was examined with anti-Flag and anti-T7, with anti-β-actin as a control of protein input. (B) Interaction of IRE1α with IKKβ in vitro. Immobilized IRE1α protein was incubated with in vitro translated IKKα, IKKβ, IKKγ, and TRAF2 (top) for 2 h and then subjected to SDS-PAGE and autoradiography (lower panel). (C) Ablation of TRAF2 impairs formation of IKK and IRE1α complex. HEK293 cells were transfected with an siRNA specific to TRAF2 and a control siRNA for 48 h and then cotransfected with T7-tagged IRE1α and Flag-tagged IKKβ for 24 h. Cell lysates were coimmunoprecipitated with anti-T7 antibody, and immunoprecipitates were probed with anti-Flag antibody. (D) ER stress induces the formation of IKK and IRE1α complex. MCF-7 cells were incubated with thapsigargin or tunicamycin for the indicated times. Cell extracts from each sample were immunoprecipitated with an anti-IKKγ antibody. Immunoprecipitates were analyzed by Western blotting with anti-IRE1α and anti-IKKβ antibodies. Five percent of cell extract from each sample was used as a control of protein input. (E) ER stress-induced NF-κB activation is impaired in TRAF2 knockdown cells. After transfection with TRAF2 siRNA and control siRNA for 48 h, MCF-7 cells were subjected to thapsigargin or tunicamycin for the indicated times. Whole-cell extracts and nuclear extracts were prepared, and IκBα degradation (top) and NF-κB DNA binding activity (bottom) were examined by Western blotting and EMSA. (F) Kinase activity is required for IRE1α to activate the IKK complex. COS-7 cells were transfected with T7-tagged wild-type and kinase-defective (K599A) mutant IRE1α. Forty-eight hours later cell extracts were prepared and protein levels of T7, IκBα, phosphorylated IκBα, and actin were detected by Western blotting. TG, thapsigargin; TU, tunicamycin; Conti, control siRNA.
FIG.2.
FIG.2.
ER stress induces formation of IRE1α and IKK complex. (A) Interaction of IRE1α with IKKβ in vivo. T7-tagged IRE1α and Flag-tagged IKKβ were cotransfected into HEK293 cells. Cell lysates were immunoprecipitated with anti-Flag antibody or T7 antibody, respectively, and then subjected to SDS-PAGE for Western blotting with anti-T7 antibody or anti-Flag antibody. Five percent of the cell extract from each sample was examined with anti-Flag and anti-T7, with anti-β-actin as a control of protein input. (B) Interaction of IRE1α with IKKβ in vitro. Immobilized IRE1α protein was incubated with in vitro translated IKKα, IKKβ, IKKγ, and TRAF2 (top) for 2 h and then subjected to SDS-PAGE and autoradiography (lower panel). (C) Ablation of TRAF2 impairs formation of IKK and IRE1α complex. HEK293 cells were transfected with an siRNA specific to TRAF2 and a control siRNA for 48 h and then cotransfected with T7-tagged IRE1α and Flag-tagged IKKβ for 24 h. Cell lysates were coimmunoprecipitated with anti-T7 antibody, and immunoprecipitates were probed with anti-Flag antibody. (D) ER stress induces the formation of IKK and IRE1α complex. MCF-7 cells were incubated with thapsigargin or tunicamycin for the indicated times. Cell extracts from each sample were immunoprecipitated with an anti-IKKγ antibody. Immunoprecipitates were analyzed by Western blotting with anti-IRE1α and anti-IKKβ antibodies. Five percent of cell extract from each sample was used as a control of protein input. (E) ER stress-induced NF-κB activation is impaired in TRAF2 knockdown cells. After transfection with TRAF2 siRNA and control siRNA for 48 h, MCF-7 cells were subjected to thapsigargin or tunicamycin for the indicated times. Whole-cell extracts and nuclear extracts were prepared, and IκBα degradation (top) and NF-κB DNA binding activity (bottom) were examined by Western blotting and EMSA. (F) Kinase activity is required for IRE1α to activate the IKK complex. COS-7 cells were transfected with T7-tagged wild-type and kinase-defective (K599A) mutant IRE1α. Forty-eight hours later cell extracts were prepared and protein levels of T7, IκBα, phosphorylated IκBα, and actin were detected by Western blotting. TG, thapsigargin; TU, tunicamycin; Conti, control siRNA.
FIG. 3.
FIG. 3.
NF-κB mediates ER stress-induced cell death. (A) Expression of mutant IκBα blocks ER stress-induced NF-κB activation. MCF-7 cells were stably transfected with pcDNA3 (empty vector) or pcDNA3 carrying an S32A and S36A double mutant HA-tagged IκBα (IκBαM MCF-7 cells). Pooled control and IκBαM MCF-7 cells were exposed to thapsigargin or tunicamycin for 2 h. NF-κB activity was measured by EMSA as described in the legend of Fig. 1. (B) Increased resistance to ER stress in IκBαM cells. Control and IκBαM MCF-7 cells were incubated with thapsigargin or tunicamycin for 30 h. Cell death was detected by MTT assay. Cell survival was expressed as absorbance relative to that of DMSO-treated controls. (C) Increased sensitivity to TNF-α- and hydrogen peroxide-induced cell death in IκBαM cells. Control and IκBαM MCF-7 cells were exposed to TNF-α (30 ng/ml) for 12 h and H2O2 (500 μM) for 18 h. Cell viability was scored by MTT assay as described in the legend of panel B. (D) Knockdown of RelA inhibits ER stress-induced cell death. MCF-7 cells were transfected with a control siRNA and an siRNA pool specific to RelA for 48 h, and cell lysates were analyzed for RelA level by Western blotting as shown at left. The same treated MCF-7 cells were subjected to treatment with thapsigargin or tunicamycin for 30 h. Cell death was detected by MTT assay (right). (E) Caspase-8 inhibitor Z-IETD-fmk decreases ER stress-induced cell death. MCF-7 cells were exposed to thapsigargin or tunicamycin with or without Z-IETD-fmk (100 μM) for 30 h. Cell death was detected by MTT assay. (F) Knockdown of caspase-8 decreases ER stress-induced cell death. MCF-7 cells were transfected with a control siRNA and an siRNA specific for caspase-8 for 48 h, and caspase-8 levels were detected by Western blotting. The same treated MCF-7 cells were subjected to thapsigargin or tunicamycin for 30 h. Cell death was detected by MTT assay. (G) ER stress activates caspase-8. Control and IκBαM MCF-7 cells were treated with thapsigargin or tunicamycin for the indicated times. Cell lysates were collected and used to measure caspase-8 activity. (H) Caspase-4 is not required for ER stress-induced caspase-8 activation. MCF-7 cells were exposed to thapsigargin or tunicamycin with or without the caspase-4 inhibitor LEVD-CHO (20 μM) for 24 h, and caspase-8 protein level was detected by Western blotting. TG, thapsigargin; TU, tunicamycin; Cont, control.
FIG. 3.
FIG. 3.
NF-κB mediates ER stress-induced cell death. (A) Expression of mutant IκBα blocks ER stress-induced NF-κB activation. MCF-7 cells were stably transfected with pcDNA3 (empty vector) or pcDNA3 carrying an S32A and S36A double mutant HA-tagged IκBα (IκBαM MCF-7 cells). Pooled control and IκBαM MCF-7 cells were exposed to thapsigargin or tunicamycin for 2 h. NF-κB activity was measured by EMSA as described in the legend of Fig. 1. (B) Increased resistance to ER stress in IκBαM cells. Control and IκBαM MCF-7 cells were incubated with thapsigargin or tunicamycin for 30 h. Cell death was detected by MTT assay. Cell survival was expressed as absorbance relative to that of DMSO-treated controls. (C) Increased sensitivity to TNF-α- and hydrogen peroxide-induced cell death in IκBαM cells. Control and IκBαM MCF-7 cells were exposed to TNF-α (30 ng/ml) for 12 h and H2O2 (500 μM) for 18 h. Cell viability was scored by MTT assay as described in the legend of panel B. (D) Knockdown of RelA inhibits ER stress-induced cell death. MCF-7 cells were transfected with a control siRNA and an siRNA pool specific to RelA for 48 h, and cell lysates were analyzed for RelA level by Western blotting as shown at left. The same treated MCF-7 cells were subjected to treatment with thapsigargin or tunicamycin for 30 h. Cell death was detected by MTT assay (right). (E) Caspase-8 inhibitor Z-IETD-fmk decreases ER stress-induced cell death. MCF-7 cells were exposed to thapsigargin or tunicamycin with or without Z-IETD-fmk (100 μM) for 30 h. Cell death was detected by MTT assay. (F) Knockdown of caspase-8 decreases ER stress-induced cell death. MCF-7 cells were transfected with a control siRNA and an siRNA specific for caspase-8 for 48 h, and caspase-8 levels were detected by Western blotting. The same treated MCF-7 cells were subjected to thapsigargin or tunicamycin for 30 h. Cell death was detected by MTT assay. (G) ER stress activates caspase-8. Control and IκBαM MCF-7 cells were treated with thapsigargin or tunicamycin for the indicated times. Cell lysates were collected and used to measure caspase-8 activity. (H) Caspase-4 is not required for ER stress-induced caspase-8 activation. MCF-7 cells were exposed to thapsigargin or tunicamycin with or without the caspase-4 inhibitor LEVD-CHO (20 μM) for 24 h, and caspase-8 protein level was detected by Western blotting. TG, thapsigargin; TU, tunicamycin; Cont, control.
FIG. 4.
FIG. 4.
ER stress induces TNF-α in an IRE1α-NF-κB dependent manner. (A) Induction of TNF-α depends on NF-κB activation. Control and IκBαM MCF-7 cells were incubated with thapsigargin or tunicamycin for the indicated times. Total RNA was analyzed for the expression of TNF-α by RT-PCR. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Detection of secreted TNF-α protein. Control and IκBαM MCF-7 cells were incubated with thapsigargin or tunicamycin for the indicated times. Supernatants of cell cultures from each sample were collected, and TNF-α protein levels were determined by enzyme-linked immunosorbent assay. Data are representative of triplicate experiments. (C) ER stress-activated TNF-α promoter activity is inhibited by mutant IκBα. Control and IκBαM MCF-7 cells were incubated with thapsigargin or tunicamycin for 2 h; then TNF-α promoter activity was detected by luciferase assay, and values were normalized based on β-galactosidase activity. (D and E) Impaired TNF-α induction in IRE1α−/− fibroblasts. Wild-type and IRE1α−/− MEFs were exposed to thapsigargin or tunicamycin for various times, as indicated. Total RNA and cell lysates, respectively, were collected and analyzed by RT-PCR for TNF-α expression and by luciferase assay for promoter activity. TG, thapsigargin; TU, tunicamycin.
FIG. 5.
FIG. 5.
ER stress-induced NF-κB activation is independent of TNFR1 signaling. (A) ER stress-induced NF-κB activation does not require de novo protein synthesis. MCF-7 cells were treated with or without CHX (5 μM) for 1 h and then with thapsigargin or tunicamycin for 2 h. Nuclear extracts were prepared for EMSA with an NF-κB probe. (B) Knockdown of TNFR1 by siRNA. MCF-7 cells were transfected with a control siRNA and a specific siRNA pool to TNFR1. Forty-eight hours after transfection, cell lysates were subjected to Western blotting with anti-TNFR1 and anti-β-actin antibodies. (C) Impaired TNF-α-induced NF-κB activation by ablation of TNFR1. After transfection with TNFR1-siRNA and control siRNA (ContsiRNA) for 48 h, MCF-7 cells were subjected to TNF-α (15 ng/ml) for 1 and 2 h. Cell extracts were collected and used to analyze protein levels of IκBα and β-actin by Western blotting with the indicated antibodies. (D and E) ER stress-induced NF-κB is independent of TNFR1 signaling. MCF-7 cells were transfected with the indicated siRNAs as described for panel C. After treatment with thapsigargin or tunicamycin for the indicated times, whole-cell extracts and nuclear extracts were prepared. Cell extracts from each sample were analyzed for IκBα levels by Western blotting. Nuclear extracts were examined by EMSA with an NF-κB probe. TG, thapsigargin; TU, tunicamycin; Conti, control siRNA.
FIG. 6.
FIG. 6.
Autocrine TNF-α contributes to NF-κB-mediated cell death in ER stress. (A) TNFR1-Fc decreases ER stress-induced cell death. MCF-7 cells were exposed to thapsigargin or tunicamycin with or without TNFR1-Fc at different concentrations for 30 h. Cell death was detected by MTT assay. Data are representative of triplicate experiments. (B) TNFR1-Fc does not affect camptothecin-induced cell death. MCF-7 cells were incubated with camptothecin (1 μM) with or without TNFR1-Fc for 24 h. Cell death was detected by MTT assay as described for panel A. (C) Fas-Fc does not influence ER stress-induced cell death. MCF-7 cells were treated with thapsigargin or tunicamycin in the presence or absence of Fas-Fc for 30 h. Cell death was detected by MTT assay as described for panel A. (D) Elimination of TNFR1 inhibits ER stress-induced cell death. After transfection with TNFR1-siRNA and control siRNA (ContsiRNA) for 48 h, MCF-7 cells were exposed to thapsigargin, tunicamycin, or camptothecin (1 μM) for indicated times. Cell death was scored by MTT assay as described for panel A. Cell survival was expressed as absorbance relative to that of DMSO-treated controls. TG, thapsigargin; TU, tunicamycin; Campt, camptothecin.
FIG.7.
FIG.7.
ER stress sensitizes cells to TNF-α-induced cell death. (A) MCF-7 cells were treated with or without thapsigargin or tunicamycin for 4 h. After being washed with medium (Dulbecco's modified Eagle medium + 10% FBS) two times, cells were incubated without or with TNF-α (30 ng/ml). Cell death was quantified by MTT assay. Data are representative of triplicate experiments. (B) ER stress inhibits TNF-α-induced activation of NF-κB. MCF-7, L929, and DU145 cells were treated with DMSO, thapsigargin, or tunicamycin for 4 h and then stimulated with TNF-α (15 ng/ml) for the indicated times. Cell lysates were prepared, and 20 μg of protein from each sample was used for Western blotting with anti-IκBα and anti-β-actin. (C) ER stress induces degradation of TRAF2. MCF-7 and L929 cells were treated with thapsigargin or tunicamycin. Cell lysates were prepared, and 20 μg of protein from each sample was used for Western blotting with anti-TNFR1, anti-TRADD, anti-RIP, anti-TRAF2, anti-IKKβ, and anti-IKKγ. (D) ER stress inhibits TNF-α-induced activation of JNK. The same samples from panel D were used for immunoblotting with anti-JNK and anti-phospho-JNK antibodies. (E) ER stress does not influence transcription of TRAF2. MCF-7 cells were treated with thapsigargin or tunicamycin for 2 and 4 h. Total RNA was collected, and the mRNA level of TRAF2 was measured by RT-PCR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) ER stress decreases stability of TRAF2. L929 cells were incubated with CHX (20 μM) or CHX plus thapsigargin (2 μM) for the indicated time, and then the TRAF2 protein level was examined by Western blotting. (G) Knockdown of TRAF2 protein sensitizes cells to ER stress-induced cell death. After transfection with TRAF2 siRNA or control siRNA for 48 h, MCF-7 cells were subjected to thapsigargin or tunicamycin for the indicated times. Cell death was assessed by an MTT assay. TG, thapsigargin; TU, tunicamycin; Conti, control siRNA.
FIG.7.
FIG.7.
ER stress sensitizes cells to TNF-α-induced cell death. (A) MCF-7 cells were treated with or without thapsigargin or tunicamycin for 4 h. After being washed with medium (Dulbecco's modified Eagle medium + 10% FBS) two times, cells were incubated without or with TNF-α (30 ng/ml). Cell death was quantified by MTT assay. Data are representative of triplicate experiments. (B) ER stress inhibits TNF-α-induced activation of NF-κB. MCF-7, L929, and DU145 cells were treated with DMSO, thapsigargin, or tunicamycin for 4 h and then stimulated with TNF-α (15 ng/ml) for the indicated times. Cell lysates were prepared, and 20 μg of protein from each sample was used for Western blotting with anti-IκBα and anti-β-actin. (C) ER stress induces degradation of TRAF2. MCF-7 and L929 cells were treated with thapsigargin or tunicamycin. Cell lysates were prepared, and 20 μg of protein from each sample was used for Western blotting with anti-TNFR1, anti-TRADD, anti-RIP, anti-TRAF2, anti-IKKβ, and anti-IKKγ. (D) ER stress inhibits TNF-α-induced activation of JNK. The same samples from panel D were used for immunoblotting with anti-JNK and anti-phospho-JNK antibodies. (E) ER stress does not influence transcription of TRAF2. MCF-7 cells were treated with thapsigargin or tunicamycin for 2 and 4 h. Total RNA was collected, and the mRNA level of TRAF2 was measured by RT-PCR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) ER stress decreases stability of TRAF2. L929 cells were incubated with CHX (20 μM) or CHX plus thapsigargin (2 μM) for the indicated time, and then the TRAF2 protein level was examined by Western blotting. (G) Knockdown of TRAF2 protein sensitizes cells to ER stress-induced cell death. After transfection with TRAF2 siRNA or control siRNA for 48 h, MCF-7 cells were subjected to thapsigargin or tunicamycin for the indicated times. Cell death was assessed by an MTT assay. TG, thapsigargin; TU, tunicamycin; Conti, control siRNA.
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