zVAD-induced autophagic cell death requires c-Src-dependent ERK and JNK activation and reactive oxygen species generation

Abstract

The treatment of L929 fibrosarcoma cells with zVAD has been shown to induce necroptosis. However, whether autophagy is involved or not in this event remains controversial. In this study, we re-examined the role of autophagy in zVAD-induced cell death in L929 cells and further elucidated the signaling pathways triggered by caspase inhibition and contributing to autophagic death. First, we found that zVAD can stimulate LC3-II formation, autophagosome and autolysosome formation, and ROS accumulation. Antioxidants, beclin 1 or Atg5 silencing, and class III PtdIns3K inhibitors all effectively blocked ROS production and cell death, suggesting ROS accumulation downstream of autophagy contributes to cell necrosis. zVAD also stimulated PARP activation, and the PARP inhibitor DPQ can reduce zVAD-induced cell death, but did not affect ROS production, suggesting the increased ROS leads to PARP activation and cell death. Notably, our data also indicated the involvement of Src-dependent JNK and ERK in zVAD-induced ROS production and autophagic death. We found caspase 8 is associated with c-Src at the resting state, and upon zVAD treatment this association was decreased and accompanied by c-Src activation. In conclusion, we confirm the autophagic death in zVAD-treated L929 cells, and define a new molecular pathway in which Src-dependent ERK and JNK activation can link a signal from caspase inhibition to autophagy, which in turn induce ROS production and PARP activation, eventually leading to necroptosis. Thus, in addition to initiating proteolytic activity for cell apoptosis, inactivated caspase 8 also functions as a signaling molecule for autophagic death.

Figure 1

zVAD induces autophagic cell death in L929 fibrosarcoma. (A) L929 cells were treated with zVAD (20 µM) for 5 or 10 h as indicated, then cells were collected and prepared for electron microscopy (TEM) analysis as described in Materials and Methods. The photos shown in (V–VIII) are the amplification of (I–IV), respectively. The arrows indicate the appearance of autophagosomes, and arrowheads indicate swelling mitochondria, which contain the double-membrane structures. (B) Cells were transfected with tfLC3 plasmid for 24 h and treated with zVAD (20 µM) or bafilomycin A₁ (BA, 100 nM), or cultured in HBSS for the indicated time. Cells were prepared as described in the method section then subjected to confocal microscopy analysis. Images are representative from three independent experiments. (C) zVAD (20 µM) and/or bafilomycin A₁ (100 nM) were added for the time periods as indicated, and total cell lysates were prepared and immunoblotted with LC3 and β-actin antibodies. Traces are representative from two independent experiments. The protein levels of LC3-I, LC3-II and β-actin were quantified by densitometry, and LC3-II /I and LC3-II /actin ratios were calculated and shown in the parentheses. (D) L929 cells were transfected with specific beclin 1 and Atg5 siRNA to knock down endogenous expression of Beclin 1 and Atg5 or nontargeting siRNA as a control. After 48 h of transfection, cells were treated with zVAD (20 µM) for 12 h, and then cell viability was measured by the MTT assay. When stimulating with indicated agents, we also collected cell lysates at the same time to determine the silencing efficiency by immunoblotting with anti-Beclin 1 and Atg5 antibodies. Data are mean ± SE M from three independent experiments. *p < 0.05, indicating significant attenuation of zVAD-induced cell death by beclin 1 siRNA. (E) After treatment with zVAD (20 µM) for 6 or 12 h, cathepsin B inhibitor (CBi, E64-d and pepstatin A, 20 µg/ml for each) or calpain inhibitor (CAi, Z-LLY-FMK, at concentration prepared in the commercial kit) for 12 h, cell lysates were prepared for determining cathepsin B and calpain activities. For the in vitro assay, zVAD, CBi or CAi were added to total lysates, and incubated for 30 min. Data are mean ± SE M from three independent experiments. *p < 0.05, indicating significant inhibition of enzyme activity.

Figure 2

ROS production from mitochondria mediates zVAD-induced cell death. (A) L929 cells were pretreated with trolox (1 mM), BHA (100 µM), DPI (1 µM), rotenone (Rot, 3 µM), FCC P (10 µM), U0126 (10 µM), SP600125 (SP, 10 µM) or SB203580 (SB, 3 µM) for 30 min, followed by zVAD (20 µM) incubation for 12 h. Cell viability was measured by the MTT assay and expressed as percentages of control. Data are mean ± SEM from three independent experiments. *p < 0.05, indicating significant attenuation of zVAD-induced cytotoxicity by trolox, BHA, rotenone, FCC P, U0126 and SP600125. (B) L929 cells were pretreated with or without BHA or SP600125 for 30 min, and then stimulated with zVAD (20 µM) for the indicated time periods. After treatment, cells were harvested and followed by intracellular ROS measurement. (C) L929 cells were pretreated with trolox (1 mM), BHA (100 µM), DPI (1 µM), U0126 (10 µM), SP600125 (10 µM) or SB203580 (3 µM) for 30 min, and then stimulated with zVAD (20 µM). After 10 h incubation, cells were harvested, followed by measurement of intracellular ROS. Data are mean ± SE M from three independent experiments. *p < 0.05, indicating significant attenuation of zVAD-induced ROS production by trolox, BHA, U0126 and SP600125. (D) zVAD (20 µM) was added for the time periods as indicated. After treatment, cells were harvested followed by measurement of mitochondrial ROS.

Figure 3

zVAD-mediated ROS production occurs downstream of autophagy, but upstream of PARP activation. (A) L929 cells were pretreated with DPQ (10 µM), 3-MA (10 mM) or wortmannin (WM, 1 µM) for 30 min, followed by zVAD (20 µM), MNNG (250 µM) or TNFα (10 ng/ml) incubation for 12 h. Cell viability was measured by the MTT assay and expressed as percentages of control. (B) Cells were pretreated with BHA (100 µM), trolox (1 mM), SP600125 (SP, 10 µM) or U0126 (10 µM) for 30 min followed by zVAD (20 µM) or with MNNG (250 µM) for the time as indicated. Total cell lysates were prepared and immunoblotted with PAR and β-actin antibodies. (C) L929 cells were pretreated with 3-MA or DPQ for 30 min, and then stimulated with zVAD (20 µM) for 10 h. After treatment, cells were harvested followed by measurement of intracellular ROS. (D) L929 cells were transfected with specific beclin 1 siRNA. After 48 h transfection, cells were treated with zVAD (20 µM) for 10 h, then intracellular ROS was measured. Data represent the mean ± SE M of three independent experiments. *p < 0.05, indicating significant attenuation of zVAD- or MNNG-induced cytotoxicity (A) and ROS production (C and D).

Figure 4

c-Src is involved in zVAD-induced autophagic cell death. (A) L929 cells were pretreated with PP2 at the concentrations indicated for 30 min, followed by zVAD (20 µM) stimulation. After 12 h incubation, cell viability was measured by the MTT assay. (B) L929 cells were pretreated with PP2 (10 µM) for 30 min, followed by zVAD (20 µM) stimulation for 10 h, and then cells were harvested for intracellular ROS measurement. (C) L929 cells were pretreated with PP2 (10 µM) for 30 min, followed by zVAD stimulation at the indicated time periods, and then cells were harvested for mitochondrial ROS measurement. The parts on the right hand show the raw data. (D) L929 cells were transfected with Src-targeted siRNA, followed by stimulation with zVAD (20 µM). Cell viability (left part) and intracellular ROS (right part) were determined. Upon stimulating with the indicated agents, cell lysates were collected to determine the silencing efficiency by immunoblotting with anti-Src antibody. Data represent the mean ± SEM of three independent experiments. *p < 0.05, indicating significant attenuation of zVAD-induced cytotoxicity and ROS production.

Figure 5

c-Src mediates JNK and ERK activation caused by zVAD. (A) L929 cells were treated with PP2 and zVAD for the indicated time periods. Cell lysates were harvested for immunobloting of JNK-p, JNK, ERK-p and ERK. (B) L929 cells were transfected with specific c-Src siRNA to knock down endogenous expression of c-Src or non-targeting siRNA as a control. After 48 h of transfection, cells were treated with zVAD (20 µM) for the indicated time. Cell lysates were harvested for immunobloting of JNK-p, JNK, ERK-p, ERK, LC3, c-Src and β-actin. (C) Cells were treated with SP600125 (SP, 10 µM) or U0126 (10 µM) for 30 min, and then stimulated with zVAD (20 µM) for the indicated time. Cell lysates were harvested for immunobloting of LC3 and β-actin.

Figure 6

zVAD induced c-Src dissociation from caspase 8 and activation. (A) After treating with zVAD (20 µM) for different periods, cell lysates were subjected to immunoprecipitation with c-Src antibody, followed by immunobloting with caspase 8 and c-Src antibodies. In (B and C), cell lysates were subjected to immunobloting with caspase 8, Tyr 418 phosphorylated Src, c-Src and β-actin antibodies.