Introduction
Bax, a pro-apoptotic protein, belongs to the Bcl-2 family of proteins [
1]. Activation of Bax plays an important role in both intrinsic and extrinsic apoptotic pathways. Activation of Bax can be achieved by binding to other Bcl-2 proteins, such as Bcl-2 or Bcl-Xl. Other Bcl-2 family proteins, such as that those contain only one Bcl-2 homology domain 3 (BH-3), can also activate Bax by directly binding to Bax [
2]. However, Bax activity is also regulated by binding to other non-Bcl-2 proteins, such as humanin, 14-3-3, and Ku70 [
3‐
5].
Using a neuroblastic type (N-type) of neuroblastoma (NB) cells, SH-SY5Y, we have established a model in which Bax activation is regulated by binding to the cytosolic Ku70 [
6]. Ku70 was originally described as an auto-antigen [
7]. However, subsequently, it was found that when dimerized with Ku80, Ku70 binds to the double strand break DNA to initiate non-homologous end-joining (NHEJ) repair [
8] process. But, in a yeast two hybrid study, Ku70 was also found to be one of the factors that bind Bax [
5]. The same study showed that cytosolic Ku70 binds to Bax and inhibits Bax’s pro-apoptotic activity. We and others have demonstrated that Ku70-Bax binding is regulated by acetylation of Ku70 such that when Ku70 is acetylated by the CREB-binding protein (CBP), Bax dissociates from Ku70 [
9,
10]. The dissociated Bax enters mitochondria resulted in release of cytochrome C that triggers cell death. Ku70 acetylation is regulated by a class II histone deacetylase (HDAC), HDAC6. HDAC6 binds and deacetylates cytosolic Ku70 such that inhibition of HDAC6, either by using class I and II HDAC inhibitors (HDACI), such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), HDAC6-specific inhibitors, tubacin, or by depleting HDAC6 using small interfering RNA (siRNA), increases cytoplasmic Ku70 acetylation that resulted in Bax dissociation [
11]. This model suggests that all cytosolic Bax binds to and is regulated by Ku70, which is inconsistent with studies demonstrating that Bax is monomeric and inactive in the cytosol [
12]. This model is based on previous studies showing that in unstimulated cells, when anti-Bax antibodies (6A7) that recognizes activated Bax are used to pull down endogenous Bax, only a small amount of Bax was immunoprecipitated. Following treatment with Bax-activating compounds, like staurosporine, 6A7 was able to pull down more Bax [
13], presumably activated Bax. Furthermore, gel filtration chromatography studies revealed that endogenous Bax is found in fractions at molecular weight corresponding to 29 kD proteins [
14]. Thus, if Bax exists as monomers in the cytosol, how does binding of Ku70 regulate Bax’s activity? What is the stoichiometry of the binding between Ku70 and Bax? Does cytosolic Ku70 bind to all Bax in the cytosol? In this study, we have addressed two questions: first, does all Bax bind to all cytosolic Ku70? Our results show that there is only a small fraction of total cytosolic Ku70 binding to a small fraction of total Bax. The majority of Bax is monomeric. However, most cytosolic Ku70 bind to other factors forming several high molecular weight complexes. There is no free or monomeric Ku70 found in the cytosol. These results suggest that other factors may also regulate Ku70-Bax binding by restricting the availability of Ku70 that can bind to Bax.
The second question is that is Ku70 a survival factor in all cells types other than the N-type NB cells? Previously, we have shown that Ku70 plays a survival role in regulating Bax pro-apoptotic activity because depletion of Ku70 in SH-SY5Y cells results in Bax-dependent cell death [
9]. However, studies have shown that, in cells, such as HeLa and HEK-293, depleting Ku70 using Ku70-specific siRNA did not induce cell death [
5,
10]. These results suggest that there may be at least two cell types in terms of Ku70 regulating Bax function: one is Ku70-depletion sensitive cells, in which Ku70 acts as a survival factor (like that in SH-SY5Y cells). These cells are sensitive to Ku70 depletion, knocking down Ku70 in these cells induces cell death. The second cell type is less sensitive to Ku70 depletion (like that in HeLa cells and HEK-293 cells), in which Ku70 is not required for survival because knocking down Ku70 in these cells does not induce cell death. Here, we provided evidence that in multiple cell types (SHEP-1, ES2, A2780, and HEK-293T cells), depletion of Ku70 does not affect cell survival. Interestingly, these cells are also less sensitive to HDACI killing compared to that of N-type NB cells. Moreover, in these cells, while HDACI treatment increases cytosolic Ku70 acetylation, Bax is neither activated nor does it dissociate from Ku70. These results suggest that there may be another mechanism that regulates Ku70-Bax formation and Bax activation in Ku70-depletion less sensitive cells.
Materials and methods
Cell culture
HEK-293T, two ovarian cancer cell lines (ES2 and A2780) and human NB cell lines SH-SY5Y, SH-EP1, GOTO, KCN-69n, and LA1-5s were cultured in modified Eagle’s minimum essential medium (MEM) supplemented with sodium pyruvate and 10 % fetal bovine serum, and maintained at 37 °C in a humidified 5 % CO2 incubator.
Cell viability assays
Cell viability was determined by either (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium reduction assay or trypan blue exclusion assay. For the MTT assay, 96-well plates were used. N-type SH-SY5Y, S-type SHEP1 human NB cell lines treated with various concentrations of SAHA, and the human ovarian cancer cell lines A2780 and ES2 were treated with varying concentrations of TSA. The viability of the cell lines was determined 24 and 48 h post-treatment by MTT assay as previously described [
15]. All experiments were carried out three times with triplicates in each experiment, and the average values and standard deviations were calculated.
Analysis of apoptosis
SH-SY5Y and HEK-293T cells grown in 60-cm plates were treated either with the solvent DMSO or with the SAHA (4 μM). Twenty-four hours after treatment, the cells were washed and suspended in annexin-binding buffer and stained with annexin V-APC and propidium iodide (PI). Induction of apoptosis was measured using a CyAn ADP Analyzer (Beckman Coulter, Inc., Indianapolis, IN) at the University of Michigan Flow Cytometry Core. The apoptotic and necrotic cells were identified based on phosphatidylserine staining with annexin on the outer leaflet of the cell membranes (apoptosis) and DNA staining by PI (necrosis and late apoptosis). Results from three independent experiments were analyzed.
For Ku70 knockdown experiments using siRNA, human NB cell lines SH-SY5Y, SH-EP1, GOTO, KCN-69n, and LA1-5s, and human ovarian cancer cell lines A2780 or ES2 cells were plated at a density of ∼2 × 106 cells per 10-cm plate 24 h before transfection. The following day, the cells were transfected either with smart pool Ku70 siRNA (silencing RNA) or with the scrambled non-targeting siRNA (Dharmacon Inc.), using nucleofector kit V (Amaxa) as per the manufacturer’s instruction. Mock transfection as well as the non-targeting siRNA transfection served as controls. The level of Ku70 was determined 72 h after transfection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using Ku70 antibodies. Either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-tubulin was used as a loading control. The viability of cells after knockdown was measured by counting cells using trypan blue exclusion analysis.
Western blot analysis
The proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane and then the blot was probed for different antibodies specific for different proteins. The following antibodies were used for western blot analyses: Ku70 (N3H10) from Santa Cruz; Ku80 (no. 2753), Bax (no. 2772S), anti-Bax antibody (6A7) (ab5714), cytochrome c oxidase subunit IV (COX IV) (no. 4844), and acetylated lysine (Ac-K-103) from Cell Signaling; and GAPDH (6C5) from Millipore and β-tubulin from Upstate. The presence of protein was visualized by using Lumigen enhanced chemiluminescence (ECL) plus PS-3.
Immunoprecipitation
Co-immunoprecipitation of Ku70, Ku80, and Bax were performed in CHAPS buffer according to the protocol described by Sawada et al. [
5] with some modifications. Cells were lysed on ice for 30 min using lysis buffer (20 mM HEPES, pH 7.5, 120 mM NaCl, and 1 % CHAPS). The extraction solution was spun twice at 3000 rpm for 10 min. The protein concentration was determined by Bio-Rad protein assay. One milligram of protein in 500 μl of lysis buffer was used for immunoprecipitation.
Subcellular fractionation: cytosolic and mitochondrial fraction
Cells were grown in 10-cm plates to 80 % confluency. The cells were washed twice with PBS, and the cells were suspended in the mitochondrial isolation buffer (5 mM HEPES pH 7.5, 210 mM Mannitol, 70 mM Sucrose, 1 mM EDTA) in 1 million cells/100 μl of buffer. The cells were swollen on ice for 30 min and were dounced in a dounce homogenizer 50 times. The lysed cells were spun at 1500 rpm for 5 min at 4 °C. The supernatant was re-spun at 3000 rpm for 10 min. The remaining supernatant, which contained cytosolic and mitochondrial fractions, was re-spun at 10,000 g for 20 min at 4 °C. The supernatant was collected as the cytosolic fraction and the pellet was collected as mitochondrial fraction. The mitochondrial pellet was washed once with the mitochondrial isolation buffer. Equal amounts of cytosolic and mitochondrial protein were separated by SDS-PAGE. The purity of the mitochondrial and cytosolic fractions was determined by immunoblotting with COX IV and β-tubulin, respectively.
DC4 cross-linking
DC4 cross-linker was used in our cross-linking analyses [
16]. Cross-linking was carried out in a buffer containing 100 mM HEPES (pH 7.5) and 150 mM NaCl. Each cross-linking reaction contained 10–15 μg of cytosolic or nuclear proteins with 0, 0.1, 0.2, 0.4, 0.8, or 1.6 mM of DC4 per sample. The cross-linking reaction was carried out on ice for 30 min. The reaction was stopped by SDS-loading buffer. Cross-linked samples were separated by 10 % SDS-PAGE, and the blot was probed with Ku70, Ku80, or Bax antibodies.
Gel filtration
Gel filtration chromatography was carried out using a Bio-Rad Biologic DueFlow system with a Superdex 200 HR 10/30 column at a flow rate of 0.5 ml per min. One milligram of cytosolic extracts was injected into the column. The running buffer was the same as the extraction buffer containing 20 mM HEPES (pH 7.5), 120 mM NaCl, and 1 % CHAPS. Half a milliliter fraction was collected. Twenty microliters of each fraction was separated by SDS-PAGE, and the blot was probed with Ku70, Ku80, or Bax antibodies.
Discussion
One of the focuses of this study was to answer a fundamental question: how much cytosolic Ku70 and Bax bind to each other in cells. A study by Sawada et al. stated that “a large proportion of the Bax population is associated with Ku70 in normal cells” [
5]. However, this statement is not consistent with the published results showing that Bax is found to be inactive and monomeric in the cytosol [
14]. In contrast, our results are consistent with this monomeric Bax model. We have shown that, in SH-SY5Y cells, which are Ku70-depletion sensitive, only a small fraction of cytosolic Ku70 binds to a small fraction of Bax. The majority of Bax is monomeric, and the majority of Ku70 is in complex with other factors, including Ku80, forming several high molecular weight complexes. Interestingly, there is no free Ku70 or monomeric Ku70 found in the cytosol.
We have proposed that Ku70 acts as a survival factor to protect the cells from dying of Bax-dependent apoptosis. How can a small amount of cytosolic Ku70 that binds to only a small amount of Bax protect the cell from dying? We believe that cells throughout life constantly receive stimuli, including death signals that affect cell viability. Some of the death stimuli may lead to apoptosis, while some of these small stimuli may only activate a few Bax molecules. As a survival mechanism, cells do not want to commit to die after receiving weak signals that activate only small amount of Bax. To survive these aberrant Bax activation signals, cells design a mechanism to block these signals. We believe that Ku70 may act as a survival factor, blocking the small amount of Bax that is being activated by weak cell death signals.
This model suggests that because there is only a small amount of Ku70 that binds to a small amount of Bax, additional Ku70 may be needed when more Bax is activated. However, where does the additional Ku70 come from if there is no free Ku70, and all remaining Ku70 that does not bind Bax is in complex with other factors? This model suggests another level of regulation of Ku70-Bax binding, in which Ku70 has to be released from other complexes to be available to bind to activated Bax. Studies have shown that Ku70 binds to several factors in the cytosol [
20‐
22]. For example, the FADD-like interleukin-1β-converting enzyme (FLICE)-inhibitory protein (FLIP) is an anti-apoptotic protein blocking caspase 8 activation by death receptors [
23]. However, FLIP, like Bax, also binds to Ku70 in an acetylation-dependent manner. FLIP binds to the Ku80-binding domain of Ku70. When Ku70 is acetylated at the same two lysines, K539 and K542, that regulate Bax binding, this will also dissociate FLIP, triggering FLIP polyubiquitination and degradation. This model suggests that Ku70 regulates apoptosis via the intrinsic pathway through Bax and the extrinsic pathway through caspase 8 [23]. Whether FLIP and Bax bind to Ku70 simultaneously and dissociate from Ku70 when Ku70 is acetylated at K539 and K542 is not clear.
Ku80 is a DNA-binding partner of Ku70 in the nucleus. Our results also show that Ku80 binds to Ku70 in the cytosol forming a higher molecular weight complex (Fig.
1b). This complex formation is not affected by HDACI treatment (Fig.
2a). However, unlike cytosolic Ku70, Ku80 does not bind Bax (Fig.
2a). These results are also consistent with the report by Sawada et al. showing that Ku80 does not bind Bax and that Ku70 does not bind Bax and Ku80, simultaneously [
5]. Whether the Ku70-Ku80 complex can regulate Ku70-Bax binding remains to be determined.
Another issue we addressed in this study is whether Ku70 that acts as a survival factor, which we established using SH-SY5Y cells, is also applicable to other cell types. Our results show that we can clearly distinguish between two cell types based on their response to Ku70 depletion: Ku70-depletion sensitive cells, in which Ku70 depletion kills cells (such as in N-type NB cells) (Fig.
3b), and Ku70-depletion less sensitive cells (such as SHEP-1, ES2, A2780, and HEK-293T cells) (Fig.
3a), in which Ku70-depletion does not kill cells.
To investigate the mechanism by which these two cell types differ in their regulation of cell death by Ku70, we treated these two cell types with HDACI. We have previously shown that, in SH-SY5Y cells, HDACI treatment induced cytosolic Ku70 acetylation and resulted in Bax release from Ku70. The released Bax translocates into mitochondria, causing cytochrome c release, triggering cell death [
17,
18]. In this study, our results show that Ku70-depletion less sensitive cells are less sensitive to HDACI-induced killing (Fig.
4c). Most importantly, cytosolic Ku70 is acetylated in these cells following HDACI treatment (Fig.
5a), but Bax is not released from Ku70. These results suggest that Ku70 acetylation regulation of Bax release in Ku70-depletion sensitive cells must be different in that in the Ku70-depletion less sensitive cells. To confirm whether Bax is activated in Ku70-depletion less sensitive cells even after HDACI-induced Ku70 acetylation, we immunoprecipitated Bax using a Bax antibody (6A7) that specifically recognizes the N-terminal of Bax when it is exposed when activated [
19]. Our results show that apoptosis and Bax activation did not occur following HDACI treatment in the Ku70-depletion less sensitive cells, while apoptotic initiation (increased in phosphatidylserine) and Bax activation were found in Ku70-depletion sensitive cells following HDACI treatment (Fig.
6a, b). Furthermore, HDACI treatment induced Bax translocation to the mitochondria in Ku70-depletion sensitive cells but not in Ku70-depletion insensitive cells (Fig.
6c). Also, cleavage of pro-caspase 3, a downstream target of Bax activation, is increased in Ku70-depletion sensitive cells following HDACI treatment but not in Ku70-depletion less sensitive cells (Fig.
6d). These results suggest that the Ku70 regulation of Bax in the Ku70-depletion less sensitive cells must be different from the conventional model, in which Bax binding to the Bax-binding domain of Ku70 is regulated by the acetylation at K539 and K542 [
10]. The two lysines are localized at the linker region of Ku70, not within the Bax-binding domain localized at the carboxyl terminal of Ku70. Acetylation of these two lysines induces a conformational change of the Bax-binding domain of Ku70, which resulted in Bax dissociation. Our model suggests that some other factors must be affecting the conformational change of Ku70 upon acetylation of these two lysines. These factors must be small because in gel filtration analyses, the pattern of Ku70 and Bax chromatograph is similar in HEK-293T cells compared to that of the SH-SY5Y cells (Figs.
1a and
4b). Another possibility is that the Ku70-depletion less sensitive cells have higher level of anti-apoptotic Bcl-2 family of proteins suppressing the activation of Bax and its association from Ku70 when Ku70 is acetylated.