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Abstract
Activation of c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is an important cellular response that modulates the outcome of the cells which are exposed to the tumor necrosis factor (TNF) or the genotoxic stress including DNA damaging agents. Although it is known that JNK is activated in response to genotoxic stress, neither the pathways to transduce signals to activate JNK nor the primary sensors of the cells that trigger the stress response have been identified. Here, we report that the receptor interacting protein (RIP), a key adaptor protein of TNF signaling, was required to activate JNK in the cells treated with certain DNA damaging agents such as adriamycin (Adr) and 1-β-D-arabinofuranosylcytosine (Ara-C) that cause slow and sustained activation, but it was not required when treated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and short wavelength UV, which causes quick and transient activation. Our findings revealed that this sustained JNK activation was not mediated by the TNF (tumor necrosis factor) receptor signaling, but it required a functional ATM (ataxia telangiectasia) activity. In addition, JNK inhibitor SP-600125 significantly blocked the Adr-induced cell death, but it did not affect the cell death induced by MNNG. These findings suggest that the sustained activation of JNK mediated by RIP plays an important role in the DNA damage-induced cell death, and that the duration of JNK activation relays a different stress response to determine the cell fate.
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Fig. 1.
Different kinetics of DNA damage-induced JNK activation in response to Adr/Ara-C and MNNG/UV-C treatment. Mouse wild-type (WT) fibroblasts were treated with 10 μg/ml of adriamycin (Adr), 50 μM 1-β-D-arabinofuranosylcytosine (Ara-C) (A) or 100 μM N-methyl-N(−nitro-N-nitrosoguanidine (MNNG), or exposed to 20 J/m2 of UV-C (B) for various times as indicated on the figure. Cell extracts were subjected to immunoprecipitation with anti- JNK1 antibody, and its activity was assayed by immune complex kinase assay with [γ-32P]ATP and GST-c-Jun (1~79) as the substrate. GST-c-Jun phosphorylation was assessed by SDS-PAGE and autoradiography (upper panel). JNK content was analyzed by Western blotting with anti-JNK antibody (lower panel).
Fig. 2.
RIP plays a role in JNK activation by Adr and Ara-C, but not by MNNG and UV-C. Wild-type (WT) and RIP-/- fibroblasts were left untreated (–) or treated with Adr (10μg/ml) and Ara-C (50μM) for 12 hrs (A), or MNNG (100μM) and exposed to UV (20 J/m2) for 30 min (B). JNK activity and expression were measured as described in the legend of Fig 1. The protein expression levels of RIP and β-actin in WT and RIP-/- fibroblasts. (C) The same amount of cell extracts from each cell line was applied to SDS-PAGE for Western blotting with anti-RIP and anti-β-actin antibodies.
Fig. 3.
Genotoxic stress-induced JNK activation is independent of TNF receptor 1 signaling. Wild-type (WT) and TNFR1-/- fibroblasts were left untreated (–) or treated with Adr (10μg/ml) and Ara-C (50μM) for 12 hrs, MNNG (100μM) for 30 min or TNF-α (15 ng/ml) for 15 min and exposed to UV (20 J/m2) for 30 min. JNK activity (top) was measured as described in the legend of Fig 1. The same cell lysates were also analyzed for the expression of TNFR1 (middle) and JNK (bottom) by Western blotting with corresponding anti-TNFR1 and anti-JNK antibodies.
Fig. 4.
ATM is required for JNK activation by Adr and Ara-C, but not by MNNG and UV-C. Wild-type (WT) and ATM-/- fibroblasts were left untreated (–) or treated with Adr (10μg/ml) and Ara-C (50μM) for 12 hrs or MNNG (100μM) for 30 min, and exposed to UV (20 J/m2) for 30 min. JNK activity and expression were measured as described in the legend of Fig. 1.
Fig. 5.
Duration of JNK activation has an important role in DNA damage-induced cell death. (A) Long term activation of JNK is associated in the Adr-induced cell death. Wild-type (WT) fibriblasts were pretreated with SP-600125 (20μM) for 30 min and then treated with various doses of Adr as indicated on the figure. At 24 hrs after treatment, the percentage of cell death was determined by trypan blue exclusion assay, as described under “Materials and Methods”. Each bar shows mean±SE of at least three independent experiments. ∗p<0.05, when compared with Adr-treated group. (B) SP-600125 abrogates the Adr-induced JNK activation. WT fibriblasts were pretreated with SP-600125 (20μM) for 30 min and treated with Adr (10μg/ml) for 12 hrs. JNK activity and expression were measured as described in the legend of Fig. 1. (C) Rapid and short term activation of JNK is not associated with the MNNG- induced cell death. (WT) fibriblasts were pretreated with SP-600125 (20μM) for 30 min and treated with various doses of MNNG as indicated on the figure. At 24 hrs after treatment, the percentage of cell death was determined as described in (A). (D) WT fibriblasts were pretreated with SP-600125 (20μM) for 30 min and then treated with MNNG (100μM) for 30 min, and JNK activity was determined as described in (B).
Fig. 6.
JNK plays as an upstream effector of DNA damage-induced PARP activation. (A) Wild-type (WT) fibriblasts were pretreated with either SP-600125 (20 μM) or PARP inhibitors (1 mM 3-AB or 30 μM DPQ). They were then treated with Adr (10 μ g/ml) for 24 hrs, and cell death was then quantified by trypan blue exclusion assay, as described in the legend of Fig 5A. Each bar shows mean ±SE of at least three independent experiments. ∗p<0.05, when compared with Adr-treated group. (B) Wild-type (WT) fibriblasts were treated with Adr (10 μ g/ml) for 12 hrs in the absence or presence of SP-600125 or PARP inhibitors (1 mM 3-AB or 30 μM DPQ), as indicated. Cell extracts were analyzed by SDS-PAGE and Western blotting with antibodies against PAR and β-actin. (C) Wild-type and RIP-/- cells were treated with Adr (10 μ g/ml) for various times, as indicated. Cell extracts were analyzed by SDS-PAGE and Western blotting with antibodies against PAR and β-actin.
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