The absence of NF-kappaB-mediated inhibition of c-Jun N-terminal kinase activation contributes to tumor necrosis factor alpha-induced apoptosis

Abstract

The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) regulates immune responses, inflammation, and programmed cell death (apoptosis). TNF-alpha exerts its biological activities by activating multiple signaling pathways, including IkappaB kinase (IKK), c-Jun N-terminal protein kinase (JNK), and caspases. IKK activation inhibits apoptosis through the transcription factor NF-kappaB, whose target genes include those that encode inhibitors of both caspases and JNK. Despite activation of the antiapoptotic IKK/NF-kappaB pathway, TNF-alpha is able to induce apoptosis in cells sensitive to it, such as human breast carcinoma MCF-7 and mouse fibroblast LM cells. The molecular mechanism underlying TNF-alpha-induced apoptosis is incompletely understood. Here we report that in TNF-alpha-sensitive cells activation of the IKK/NF-kappaB pathway fails to block TNF-alpha-induced apoptosis, although its inactivation still promotes TNF-alpha-induced apoptosis. Interestingly, TNF-alpha-induced apoptosis is suppressed by inhibition of the JNK pathway but promoted by its activation. Furthermore, activation of JNK by TNF-alpha was transient in TNF-alpha-insensitive cells but prolonged in sensitive cells. Conversion of JNK activation from prolonged to transient suppressed TNF-alpha-induced apoptosis. Thus, absence of NF-kappaB-mediated inhibition of JNK activation contributes to TNF-alpha-induced apoptosis.

FIG. 1.

Activation of NF-κB is insufficient to suppress TNF-α-induced apoptosis in MCF-7 cells. (A) MCF-7 cells were transfected with an expression vector encoding wild-type HA-IKKβ (WT) or HA-IKKβ(EE) (1.5 μg of each). After 48 h, cells were treated with or without TNF-α (20 ng/ml) for 15 min. The activity of wild-type HA-IKKβ or HA-IKKβ(EE) was measured by immune complex kinase assays (KA) with GST-IκBα as the substrate, and their expression was analyzed by immunoblotting with anti-HA antibody (IB). (B) MCF-7 or MCF-7-R cells were transfected with the 2× NF-κB-Luc reporter plasmid (0.2 μg each), along with expression vectors encoding either HA-IKKβ(EE), HA-RelA, or an empty vector (0.4 μg of each). After 30 h, cells were treated with or without TNF-α (20 ng/ml) for 8 h. Relative luciferase (LUC) activity was determined as previously described (28). The results are presented as means ± standard errors and represent three separate experiments done in duplicate. (C and D) MCF-7 (C) or MCF-7-R (D) cells were transfected with expression vectors encoding HA-IKKβ(EE), HA-RelA, or an empty vector (2.0 μg of each), along with the transfection marker GFP (0.5 μg). After 48 h, cells were treated with or without TNF-α (20 ng/ml) for 11 h (C) or treated with or without TNF-α (20 ng/ml) plus CHX (10 μg/ml) or CHX alone for 6 h (D). Cells were stained with Hoechst, and nuclear condensation was visualized by fluorescence microscopy. The apoptotic death of transfected (GFP-positive) cells was calculated by counting eight randomly selected areas (total, 40 to 50 cells per area). The results are presented as means ± standard errors and represent three separate experiments.

FIG. 2.

TPA, but not IL-1, inhibits TNF-α-induced apoptosis in MCF-7 cells. (A) MCF-7 cells were treated with or without TNF-α (20 ng/ml) for 15 min, TPA (100 ng/ml) for 30 min, or IL-1 (5 ng/ml) for 15 min. IKK activity and expression were examined as described in the legend to Fig. 1. (B) MCF-7 cells were transfected with the 2× NF-κB-Luc reporter plasmid (0.2 μg). After 30 h, cells were treated with or without TNF-α (20 ng/ml), TPA (1.0 ng/ml), or IL-1 (5 ng/ml) for 8 h. Luciferase (LUC) activity was determined as previously described (28). The results are presented as means ± standard errors and represent three separate experiments done in duplicate. (C) MCF-7 cells were pretreated with TPA (100 ng/ml) or IL-1 (5 ng/ml) for 3 h and then stimulated with TNF-α (20 ng/ml) for 11 h. Apoptotic cell death was calculated by counting six to eight randomly selected areas (total, 150 to 200 cells per area) as described in the legend to Fig. 1. The results shown represent three separate experiments. (D) MCF-7 cells were pretreated with or without TPA (1.0 ng/ml) or IL-1 (5 ng/ml) for 3 h and then stimulated with TNF-α (20 ng/ml) for 11 h. Caspase (Casp) activity was measured with DEVD-AFC as the substrate in accordance with the manufacturer's (Clontech) manual. Caspase 7 activity is presented as means ± standard errors and represents three separate experiments. (E) MCF-7-R cells were pretreated with or without IL-1 (5 ng/ml) for 3 h and then stimulated with TNF-α (20 ng/ml)-CHX (10 μg/ml) for 10 h. Apoptotic cell death was calculated as described for panel C, and the results shown represent three separate experiments. (F) MCF-7-R cells were pretreated with or without IL-1 (5 ng/ml) for 3 h and then stimulated with TNF-α (20 ng/ml) and CHX (10 μg/ml) for 6 h. Caspase activity was measured as described for panel D.

FIG. 3.

Inhibition of the IKK/NF-κB pathway promotes TNF-α-induced apoptosis in MCF-7 cells. (A) MCF-7 cells were pretreated with or without NBD at various doses for 3 h and stimulated with TNF-α (20 ng/ml) for 15 min. IKK activity and expression were measured as described in the legend to Fig. 2. (B) MCF-7 cells were pretreated with NBD (150 μM) for 3 h and stimulated with TNF-α (20 ng/ml) for 11 h. Cells were Hoechst stained, and apoptotic cells were calculated as described in the legend to Fig. 2C. The results are presented as means ± standard errors and represent three separate experiments. (C) MCF-7 cells were pretreated with ALLN (150 μM) for 2 h and stimulated with TNF-α (20 ng/ml) for 15 min. Degradation of IκBα proteins was analyzed by immunoblotting with anti-IκBα antibody. (D) MCF-7 cells were transfected with the 2× NF-κB-Luc reporter plasmid (0.2 μg). After 30 h, cells were pretreated with or without ALLN (150 μM) for 2 h and stimulated with TNF-α (20 ng/ml) for 8 h or left untreated. Cells were harvested, and relative luciferase (LUC) activity was determined. The results are presented as means ± standard errors and represent three separate experiments done in duplicate. (E) MCF-7 cells were pretreated with ALLN (150 μM) for 2 h and stimulated with TNF-α (20 ng/ml) for 11 h. Apoptotic cell death was calculated as described for panel B.

FIG. 4.

JNK activation is involved in TNF-α killing of MCF-7 cells. (A) MCF-7 cells were pretreated with SP600125 (20 μM) for 30 min and stimulated with TNF-α (20 ng/ml) for 15 min. JNK was immunoprecipitated with anti-JNK1 antibody, and its activity was measured by immune complex kinase assay with GST-c-Jun(1-79) as the substrate. JNK content was analyzed by immunoblotting with anti-JNK antibody. The same cell lysates were also analyzed for activation of p38 or ERK by immunoblotting with corresponding anti-phospho antibodies. (B) MCF-7 cells were pretreated with SP600125 (SP; 20 μM) for 30 min and stimulated with TNF-α (20 ng/ml) for 11 h. Apoptotic cell death was calculated as described in the legend to Fig. 2C. (C) MCF-7 cells were cotransfected with expression vectors encoding GFP (0.5 μg) and either the HA-JNKK2-JNK1 fusion protein, the dominant negative mutant HA-JNKK2(K149M), or an empty vector (2.0 μg of each). Cells were treated with or without TNF-α (20 ng/ml) for 11 h and stained with Hoechst. The death of transfected (GFP-positive) cells was calculated as described in the legend to Fig. 1.

FIG. 5.

JNK phosphatase(s) mediates the inhibitory effect of TPA on JNK activation in MCF-7 cells. (A and B) MCF-7 cells were pretreated with IL-1 (5 ng/ml) or TPA (100 ng/ml) for 1 h and stimulated with TNF-α (20 ng/ml) for 15 min. JNK activity and expression was measured as described in the legend to Fig. 4. (C and D) MCF-7 cells were pretreated with OV (100 μM) for 30 min prior to treatment with TPA (100 ng/ml) for 1 h. Cells were stimulated with TNF-α (20 ng/ml) for either 15 min (C) or 11 h (D). JNK activity and expression were measured as described in the legend to Fig. 4, while apoptotic cell death was detected and calculated as described in the legend to Fig. 1. The results shown represent three separate experiments.

FIG. 6.

Prolonged JNK activation by TNF-α in MCF-7 cells is due to absence of NF-κB-mediated inhibition. (A and E) MCF-7-R or MCF-7 cells were treated with TNF-α (20 ng/ml) for various times as indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (B and F) MCF-7-R or MCF-7 cells were infected with Ad/IκBα(A32/36) (MOI, 500). After 24 h, cells were treated with TNF-α (20 ng/ml) for various times as indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (C, D, G, and H) MCF-7-R or MCF-7 cells were transfected with an expression vector encoding HA-XIAP or HA-RelA or with an empty vector (1 μg of each), along with M2-JNK (0.5 μg). After 48 h, cells were treated with TNF-α (20 ng/ml) for various times as indicated. JNK activity was measured as described in the legend to Fig. 4. (H) M2-JNK1 was immunoprecipitated from extracts from vector- and HA-XIAP-transfected cells that had been normalized to contain the same amount of M2-JNK1 protein. Expression of M2-JNK, HA-RelA, and HA-XIAP was analyzed by immunoblotting with anti-M2 or anti-HA antibody.

FIG. 7.

Prolonged JNK activation contributes to TNF-α-induced apoptosis in MCF-7 cells. (A) MCF-7 cells were stimulated with TNF-α (20 ng/ml). After 30 min, cells were treated with SP600125 (20 μM) and harvested at the time points indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (B) MCF-7 cells were treated with TNF-α (20 ng/ml) and 30 min later were treated with or without SP600125 (20 μM). After 11 h, cells were stained with Hoechst and visualized by fluorescence microscopy. Cell death was calculated as described in the legend to Fig. 2C. (C) MCF-7-R cells infected with Ad/IκBα(A32/36) (MOI, 500) were treated with TNF-α (20 ng/ml). After 30 min, cells were treated with SP600125 (20 μM). JNK activity and expression were measured as described in the legend to Fig. 4. (D) MCF-7-R cells were infected with Ad/IκBα(A32/36) or Ad/GFP (MOI, 500) and treated with TNF-α (20 ng/ml). After 30 min, cells were treated with SP600125 (20 μM) and cell death was calculated 7 h later as described in Fig. 2C. The results shown represent three separate experiments.

FIG. 8.

Prolonged JNK activation affects the susceptibility of cells to TNF-α-induced apoptosis. (A) TNF-α-sensitive LM cells were treated with TNF-α (20 ng/ml) and harvested at the time points indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (B) LM cells were cotransfected with expression vectors encoding M2-JNK (1.5 μg) and either the JNKK2(K149M) mutant or an empty vector (6.0 μg of each). Cells were treated with or without TNF-α (20 ng/ml) for 15 min. The activity of M2-JNK and the expression of transfected constructs were determined as previously described (49). (C) LM cells were cotransfected with expression vectors encoding GFP (1.5 μg) and either the JNKK2(K149M) mutant or an empty vector (6.0 μg of each). Cells were treated with or without TNF-α (20 ng/ml) for 8 h, stained with Hoechst dye, and visualized by fluorescence microscopy. The death rate of transfected (GFP-positive) cells was calculated by counting six randomly selected areas (total, 30 to 40 cells per area). Results are presented as means ± standard errors and represent two individual experiments. (D) TNF-α-insensitive HT-1080 cells were treated with TNF-α (20 ng/ml) for various times as indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (E) HT-1080 cells were infected with Ad/IκBα(A32/36) (MOI, 500). After 24 h, cells were treated with TNF-α (20 ng/ml) for various times as indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (F) HT-1080 cells infected with Ad/IκBα(A32/36) (MOI, 500) were stimulated with TNF-α (20 ng/ml). After 30 min, cells were treated with SP600125 (20 μM) and harvested at the time points indicated. JNK activity and expression were measured as described in the legend to Fig. 4. (G) HT-1080 cells were infected with Ad/IκBα(A32/36) or Ad/GFP (MOI, 500) and treated with TNF-α (20 ng/ml). After 30 min, cells were treated with or without SP600125 (20 μM) and cell death was calculated 9 h later as described in the legend to Fig. 2C. The results shown represent three separate experiments.