Introduction
As one member of activator protein-1 (AP-1) transcription factor, a growing number of transcription dependent and independent functions have been described, while attesting to its capacity to regulate diverse and often opposing functions, which implicated some of the most challenging therapeutic targets [
1,
2]. Growing evidence indicates that a cytoplasmic localization of activating transcription factor 2 (ATF2) is associated with cell death in disease or cellular stress states [
3]. Following ionizing irradiation with prostate cancer cells, ATF2 had been observed to accumulate in the cytoplasm [
4]. Furthermore, the forced expression of cytoplasmic ATF2 peptides induces melanoma cell death and thereby reduces the transcriptional activity of endogenous ATF2 [
5]. Further studies have shown that genotoxic stress induces translocation of nuclear ATF2 to the cytoplasm, which coincides with the reduced transcriptional activity of ATF2 [
6,
7].
Because ATF2 localizes at the mitochondrial outer membrane result in reduced mitochondrial membrane potential, with concomitant leakage from the mitochondria, hallmarks of mitochondrial-dependent cell death. Hence, ATF2 recruitment to the mitochondria is associated with its tumor suppressor activities. Despite the fact that functional studies of ATF2 provide no compelling reason for decreasing HK1 bind to VDAC1 [
8,
9], possible mechanism might be formation of VDAC channel with some unknown factors larger than that of the individual proteins to mediate cytochrome c release. ATF2 also reported play an important role in the conformation and thus activation of Bax [
1]. Conformational changes and homo-oligomerization are two critical events associated with the activation of BAX/BAK by BH3s and VDAC1. However, the underlying mechanisms remain unsettled. It is especially complex for BAX due to its change in the subcellular localization during apoptosis. An understanding of the precise mechanisms underlying its function at the mitochondria, could provide a platform for identifying small molecues that affect the involved signaling. Similarly, it will be important to determine its mitochondrial function and their possible relationship to the Bcl-2 family protein signaling that is associated with mitochondrial-based death programs [
10,
11]. Indeed, evidence involved in activator BHS-only proteins (BH3s), such as Bim, has been presented, which could induce BAX and VDAC reconstituted into liposomes, forming a new channel at the MOM [
12-
14]. According to this facts, we can speculated that there is possible signal relationship with BH3s and ATF2, which is related to mitochondrial involved apoptosis.
Here, we investigate the role of ATF2 in mitochondrial apoptosis and the mechanisms underlying the interaction of ATF2 with other proapoptosis proteins. Our work revealed that, at the membrane of mitochondrial, the hierarchical involvement of Bim, VDAC1, and ATF2 break the hexokinase-1 (HK1) and voltage-dependent anion channel-1 (VDAC1) complexes, thereby enhancing mitochondrial permeability and releasing cytochrome c. The exploration of the mechanisms for ATF2 mitochondrial localization offers a theoretical basis to guide cancer cells to apoptosis.
Methods
Cell culture, antibodies, regents, and drugs
The tumor cell lines B16F10, A549, EG7, and LL2 were selected for this research. These cells were gifts of Prof. Hongbo Luo, Harvard University, and were maintained in RPMI 1640 (Gibco, Gaithersburg, USA) or DMEM (Life Technologies, CA, USA), each supplemented with 10% fetal bovine serum and penicillin G sodium and streptomycin sulfate (100 units/ml) in a humidified atmosphere of 5% CO2 and 95% air. ABT-737 (Selleckchem, Souffelweyersheim, France) used at 1 μM final concentration. MitoTracker Red CMXRos (M7512) and leptomycin B (LMB) (sc-358688) were purchased from Invitrogen (Eugene, OR, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. All antibodies including ATF2(C-19, sc-187), p-ATF2(F-1, sc-8398), COX4 (D-20, sc-69359), Bim (N-20, sc-8265), PUMAα (N-19, sc-19187), Bax (P-19, sc-526), cytochrome c (C-20, sc-8385), VDAC1 (B-6, sc-390996), Mcl-1 (S-19, sc-819), β-Actin (C4, sc-47778) were purchased from Santa Cruz Biotechnology. The monoclonal anti-HK1(C35C4, 2024) antibody was obtained from Cell Signaling Technologies (MA, USA). Paclitaxel ((PTX, 33069-62-4) was obtained from Beijing Zhongshuo Pharmaceutical T & D Co., Ltd (Beijing, China). Staurosporine (STS), cisplatin, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), carbonyl cyanide m-chlorophenylhydrazone (CCCP), and propidium iodide were purchased from Sigma (St. Louis, MO, USA). The annexin V-FITC kit (K201-100) was purchased from Biovision. PTX was purchased from Calbiochem (La Jolla, CA) and dissolved in 100% dimethyl sulfoxide to produce a stock solution of 1.0 mM.
RNA interference
Cells were transfected at ~70-90% confluence (approximately 1 × 10
5 cells/ml density). ATF2-specific shRNA clones (ID: TRCN0000013713, Sigma, USA) were obtained from Open Biosystems (catalog no. RHS4533). Lentiviral particles packaged with ATF2 shRNA or scrambled shRNA (control) were generated and spin infected into the target cells in the presence of 10 mg/ml polybrene (Sigma, USA) [
15]. The transfection of a synthetic siRNA (25 nM) for VDAC1 was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The sense sequence of the double-stranded siVDAC1 siRNA was 5′ AGUGACGGGCAGUCUGGAATT 3′. A scrambled siRNA, which served as a negative control, was purchased from GenePharma (Shanghai, China). HK1 siRNAs (ID: 1599) were obtained from Ambion (USA). The same siRNA reagents were added to the medium at 24 hours post-transfection for 24 hours. The western blot analysis was used to evaluate gene silencing effects. The appropriate controls were included during the entire siRNA knockdown process, confirming the specificity of the siRNA. For the construction of tetracycline-regulated gene expression vectors expressing ATF2
T52A mutants (mitochondria localization), DNA was amplified by PCR using pEF-HA-ATF2 (WT, T52A mutants), a gift of Ze’ev A. Ronai (Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA), and subcloned into pTHE, resulting in pTHE-WT, ATF2
T52A, which were transfected into B16F10 and screened with tetracycline.
Cell viability determination (MTT Assay)
Subconfluent monolayers of the B16 cell line were established in 96-well plates. After overnight incubation, the cells were exposed to different treatments in a medium containing 0.5% fetal bovine serum for 72 hours; 10-μl aliquots of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) solution (10 mg/mL in PBS) were added, followed by 100 μL of 10% sodium dodecylsulfate (SDS) to dissolve the formazan crystals formed. The absorption of the samples was determined using an ELISA reader (Anthos Mikrosysteme GmBH, Germany) at a wavelength of 570 nm. A standard optical density of the untreated control cells was considered at 100% viability. Survival was evaluated by the absorbance of the treated cells normalized to the controls.
Confocal immunofluorescence assays
Cells from different treatment groups were incubated with MitoTracker Red (25 nM) for 15 minutes, fixed, permeabilized, and stained with antibodies for the detection of ATF2 (20 F1). The primary antibodies were revealed using either goat anti-rabbit or anti-mouse IgG conjugated to Alexa 488 (green) (1:500, diluted in blocking solution) from Molecular Probes-Invitrogen. After 1 hours of incubation, the slides were mounted, and the stained cells were analyzed using a confocal microscope (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany).
Apoptosis assay by annexin V/propidium iodide staining
At various time points, control and treated cells were collected following treatment and subjected to apoptosis measurement using the annexin V/propidium iodide (PI) detection kit (R&D Systems) according to the manufacturer’s instructions. A total of 10,000 cells (within whole-cell gates) per replica (3 independent experiments) were subjected to a flow cytometric analysis to evaluate the green fluorescence of annexin V and the red fluorescence of DNA-bound PI. All the data were analyzed with FlowJo software (TreeStar, OR).
Cytochrome c release assay
Isolated tumor cells (5 × 107) were collected and assayed with the Cytochrome c Apoptosis Assay Kit (Cat. #K257-100, Biovision, CA, USA). Briefly, the cells were homogenized with the cytosol extraction buffer provided in the kit and then centrifuged at 700 × g for 10 minutes at 4°C to remove the debris. The supernatant was then centrifuged at 10,000 × g for 30 minutes at 4°C; the pellet contained the mitochondrial fraction, and the supernatant was collected as the cytosolic fraction. These fractions were analyzed for cytochrome c by western blotting using the cytochrome c antibody provided in the kit.
Immunoprecipitation and analysis of protein expression
Cells, transfected as indicated, were lysed in the buffer for 45 min. Lysate aliquots of equal concentration were then incubated overnight with 2 μg of anti-ATF2, −VDC1, −Bim, and -Puma antibodies in an overhead rotator, followed by 20 μl protein G-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden) for 2 h. The immunoprecipitated proteins were incubated at 70°C for 15 min and analyzed by immunoblotting with conformation-specific primary antibodies against ATF2, VDC1, Bim, Puma, HK1, and VDAC1 (Cell Signaling Technology). β-actin (Chemicon International, Temecula, CA, USA) was performed as loading control.
Cell fractionation
Fractions of cytoplasm nuclear, and mitochondria were separated using a commercial Qproteome mitochondria extraction kit and a Qproteome nucleus extraction kit (Qiagen, Toronto, ON, Canada). Briefly, cells were firstly lysed and centrifuged for 5 min at 1000 × g to remove unbroken cells and nuclei. The supernatant was separated from the pellet and centrifuged at 2,200 × g for 20 min at 4°C to pellet the mitochondria-enriched heavy membrane fraction. The resulting supernatants were combined and further centrifuged at 4°C at 12,000 × g for 30 min at 4°C to obtain the cytoplasmic fraction. An immunoblot analysis was performed as described below.
Western blot analysis
Cells from different treatment groups were lysed using a protein extraction buffer. Total proteins (10 μg) were separated by SDS-PAGE and transferred to nylon membranes (Shanghai Sangon Biotech, Shanghai, China). The blots were hybridized with antibodies indicated above. The secondary antibody, horseradish peroxidase-coupled immunoglobulin (Jingmei Biotech Co., Ltd. Shenzhen, China), was then inculated for 1 h. β-actin (Sigma) was used as loading control. All critical blots and immunoprecipitation experiments were repeated at least three times.
Mitochondrial membrane potential detection
Cells were treated and resuspended in serum-free medium at a concentration of 1 million cells/ml. Each sample was added 5 μl of JC-1 dye (200 μM) for incubation at 37°C, 30 min. The samples were measured by flow cytometry, with 10,000 events collecting. Results were also observed under fluorescence microscopy.
Tumor implantation procedure
C57BL/6 female (8–10 weeks old) mice were purchased from Chongqing Medical University Animal Center (Chongqing, China). All animal experiments were performed with the approval of the Animal Institute Committee. B16F10 cells stably transfected with ATF2 shRNA, ATF2T52A or with empty vector (1.0 × 106/0.1 ml) were injected subcutaneously. The tumor sizes were evaluated using calipers every 2 to 3 days, and the tumor volumes were calculated using the formula: volume = (a2 × b)/2 (a, the short tumor length; b,the long tumor length). In one arm of the experiment, nonnecrotic, single-cell suspensions from tumor tissue were prepared for FACS staining of annexin V/propidium iodide. A portion of the freshly isolated tumor tissue was subjected to a western blotting assay and real-time PCR analysis, as described in the results section.
Statistical analysis
Data are expressed as means ± standard errors of the mean (SEM). Unless indicated otherwise, comparisons were determined using the Student’s t test and one-way ANOVA. P < 0.05 were considered as significance difference.
Discussion
The cytosolic localization of ATF2 has been associated with tumor suppressor activity in some solid tumors [
7,
16-
20]. However, targeting this pro-apoptotic protein for tumor treatment requires knowledge and understanding of their modes of action. Several possible mechanisms of action have been proposed in the previous reports. The present work offer insight into the mechanism by which mitochondrial ATF2 might help to cytotoxic stress induced mitochondrial membrane permeabilization (MMP). While the ATF2 still localizes to mitochondria and functions normally, releases of cytochrome
c and apoptotic cell death were induced. Mitochondrial ATF2 impairs HK1/VDAC1 complexes and mitochondrial membrane integrity, sensitizing cells to apoptosis. Moreover, we demonstrated that apoptosis induction hierarchically involves Bim and VDAC1. The proapoptotic protein Bim was shown to promote apoptosis through interactions with VDAC1 and ATF2. Finally, a decrease in ATF2 mitochondrial expression led to enhanced tumorigenesis ability in vivo.
ATF2 appears to be a convergence point for a variety of cellular survival and death signals. Considering that the major cellular site targeted by mitochondrial ATF2 is the mitochondrion
, several mechanisms were proposed to explain the action of such proteins at the mitochondria [
1]. In accordance with previous results [
3], genotoxic stress in melanoma cells was found to induce the predominant mitochondrial localization of ATF2 and promote cell death. The overexpression of mitochondrial ATF2 induced mitochondrial depolarization, a phenomenon associated with apoptosis, regardless of the cellular host. We also indicated that ATF2 depletion cells have a lower capacity to induce apoptosis in response to genotoxic stress, suggesting that lying in the mitochondrial outer membrane (MOM), ATF2 is a conserved mitochondrial element of the death machinery. These findings are consistent with previous results that the cytosolic ATF2 is necessary for mitochondria-mediated apoptosis (29). ATF2 is required for cytochrome
c release when induced by various stimuli. Previous results indicated that the cytosolic ATF2 is necessary for mitochondria-mediated apoptosis [
15], consistent with current findings. Cytochrome
c release also found to ATF2 requirement with various stimuli exposure. Indeed, apoptosis induced of by PTX, cisplatin, and STS, acting on multiple pathways, all ultimately led to cytochrome
c release in cells expressing native ATF2.
It has been shown that ATF2 might regulate and control the availability of VDAC1 to HK1, Bcl-2, and Bcl-xL [
3]. Herein, we clearly show that ATF2 can interact with VDAC1 to disrupt the HK1/VDAC complex in various cancer cells, suggesting that ATF2 might bind to VDAC1 in competing with HK1 and thus promote cytochrome
c release.
It has also been suggested that the proapoptotic Bim protein induces conformational changes in BAX to promote the targeting and homo-oligomerization of BAX at the MOM [
21]. The proapoptotic proteins BAX and BAK were proposed to form heterodimeric complexes, with physical associations between VDAC1 and BAX being reported in various experimental models [
22]. Here, we not only demonstrated a direct interaction of Bim with VDAC1 but also showed the function of this interaction in mediating the proapoptotic activity of ATF2 to modulate the interaction of VDAC1 and BAX. Our results point to a role for ATF2 as an activator of Bim. Serial siRNA experiments and epistatic analyses suggest that ATF2triggers the conformational activation of Bim, whereas, VDAC1 activation (downstream of ATF2 signal) need the Bim expression. Accordingly, VDAC1 does not adopt its active conformation when Bim is absent, whereas ATF2 remains inactive. Furthermore, pharmacological inhibition of Bcl-2/Bcl-X
L with the BH3 mimetic ABT-737, was able to rescue the defect of VDAC1 inactivation triggered by ATF2 deficient. It is worth noting that in the conditions used here, ABT-737 was not able to induce VDAC1 activation per se.
Therefore, our results define the pathway by which ATF2, Bim, and VDAC1 hierarchically operate together, which has not been reported to date. Although it is known that ATF2 can function upstream of Bim, at least in some paradigms of apoptosis, it has never been indicated that Bim might seat in a intermediate position between ATF2 and VDAC1, as present here. Furthermore, BAX and VDAC1 were reported to assemble amultimeric channel, large enough for the release of proteins [
23-
25]. Similarly, the biophysical interactions between Bim, ATF2, and VDAC1 might be important cytochrome c release. The requirement of ATF2 for cytochrome c release and its functional interaction with Bim indicate ATF2 as being critical factor in mediatting cell death. Future work must address the biochemical mechanisms underlying the cooperation between Bim and VDAC1. Interestingly, it has been shown that Bax and VDAC1 can form a large, multimeric channel that allows for the release of proteins. Hence, it will be important to investigate the putative physical and functional interactions between Bim, Bax and VDAC1 in the context of PTX-triggered cell death.
Concerning the function of ATF2 on tumorigenesis,we detected increased proliferation in tumors from an ATF2-deficient B16F10 cell line injected into C57BL/6 mice, in accordance with what occurs on apoptosis in ATF2-depletion cells. In fact, the mice that displayed an increased growth of B16F10 cells also showed a lower expression of ATF2 in the tumor tissue. This dominance has been detected in several other tumors [
26,
27]. Hence, we believe that the ATF2 may be more important in promoting tumor growth in this model, and it can be speculated that ATF2 might initially contribute to the events towards tumor proliferation.
Acknowledgements
1. We thank Prof. Xianqing Jin for providing technical assistance and insightful discussions during the reparation of the manuscript. We thank Dr. Xiaoyong Zhang, of the Wistar Institute, USA, for help with the language revision of the manuscript.
2.This research was supported by National Natural Science Foundation of China (No: 30973440, 30770950), Key project of National Natural Science Foundation of China (NO.30330590), and Key project of Chongqing Natural Science Foundation (CSTC, 2008BA0021).
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CL designed and performed the experiments, analyzed the data, and prepared the manuscript. QL helped with designing the experiments, analyzing the data, and evaluating the manuscript. CL bred the mice. CG designed the experiments, analyzed the data, and wrote the paper. All authors read and approved the final manuscript.