Abstract

The nuclear factor-κB (NF-κB) family of transcription factors can directly or indirectly regulate many important areas of biology, including immunity, inflammation and cell survival. One intriguing aspect of NF-κB crosstalk with other cell signalling pathways is its regulation of mitochondrial biology, including biogenesis, metabolism and apoptosis. In addition to regulating the expression of mitochondrial genes encoded in the nucleus, NF-κB signalling components are also found within mitochondria themselves and associated with mitochondrial DNA. However, complete biochemical analysis of mitochondrial and sub-mitochondrial localisation of all NF-κB subunits has not been undertaken. Here, we show that only the RelA NF-κB subunit and its inhibitor IκBα reside within mitochondria, whilst p50 is found in the endoplasmic reticulum (ER). Fractionation of mitochondria revealed that only RelA was found in the mitoplast, the location of the mtDNA. We demonstrate that hypoxia leads to a very rapid but transient accumulation of RelA and IκBα in mitochondria. This effect required reactive oxygen species (ROS) but was not dependent on the hypoxia sensing transcription factor subunit HIF1α or intracellular Ca2+ release. We also observed rapid mitochondrial localisation of transcription factor STAT3 following hypoxia. Inhibition of STAT3 blocked RelA and IκBα mitochondrial localisation revealing a previously unknown aspect of crosstalk between these key cellular regulators.

Introduction

The nuclear factor-κB (NF-κB) family of transcription factors regulate the expression of a vast array of genes in response to extra- and intracellular stimuli by binding to κB DNA responsive elements within their promoters [1]. An important regulator of immunity, inflammation, development, cell division and survival, NF-κB also participates in many additional processes via crosstalk with other signalling pathways [2]. Aberrant NF-κB activation is therefore associated with many diseases, including cancer, with tumour cells often exhibiting constitutively active NF-κB [3].

Mammalian NF-κB consists of five subunits, RelA (p65), RelB, c-Rel, p100/p52 and p105/p50, all of which contain the REL Homology Domain (RHD) that mediates DNA binding and dimer formation [4,5]. The C-termini of RelA, RelB and c-Rel also contain transactivation domains, essential for controlling the expression of target genes [6,7]. NF-κB subunits form homo or heterodimers, held in the cytoplasm in an inactivated state through interactions masking their nuclear localisation sequences. RelA and c-Rel are inactivated by the ankyrin repeat containing Inhibitor of NF-κB (IκB) proteins such as IκBα [8]. The NF-κB subunit precursors p100 and p105 have their own C-terminal ankyrin repeat domains and can also function as inhibitors [9]. Activation of NF-κB therefore requires the removal of these inhibitors, either via degradation of the IκB proteins or processing of p100 and p105 to the shorter forms p52 and p50, respectively [10–12]. IκBs are phosphorylated and targeted for proteasomal degradation via the IκB kinase (IKK) complex, consisting of IKKα, IKKβ and NEMO [13]. This allows the nuclear localisation of the active NF-κB complexes, followed by target gene promoter binding and transcriptional regulation. It is now emerging that apart from their functions in NF-κB signalling, components of this pathway also play other independent roles [14,15].

Mitochondria are double membrane organelles containing their own genetic material encoded on circular DNA molecules (mtDNA) that are essential for energy, metabolite and reactive oxygen species (ROS) production, as well as calcium storage and apoptosis [16,17]. Mitochondrial dysfunction can lead to many diseases and is a contributing factor to ageing and cancer development. NF-κB signalling can directly regulate at least three aspects of mitochondrial biology: biogenesis by controlling the levels of dynamin-like protein Opa1 required for mitochondrial fusion [18]; metabolism by regulating the expression of a number of mitochondrial genes encoded in the nucleus as well as the mtDNA [19–22]; and apoptosis [19,21–24]. In most cases, mitochondrial changes induced by NF-κB culminate in cell survival. Changes in mitochondrial metabolism through NF-κB activity could also contribute towards its tumour promotor function via suppression of p53 signalling [19,23].

RelA, p50 and IκBα have all been reported to reside within the mitochondria of cancer cell lines as well as in tissues [20–27], suggesting that some NF-κB effects on mitochondria might be direct. Mitochondrial RelA was found to associate with the D-loop region of mtDNA and alter cytochrome b (CYTB) expression but this was still observed in a DNA binding mutant form of RelA, suggesting it is likely to be an indirect interaction [20,21]. Additionally, analysis of publically available ENCODE chromatin immunoprecipitation (ChIP)-seq datasets of nuclear transcription factor occupancy on mtDNA also suggests that NF-κB does not associate directly with the mitochondrial genome [28]. Therefore, it is likely that mitochondrial NF-κB does not have a classical transcriptional role, but rather, alters gene expression of mtDNA encoded genes as a co-factor or through alternative mechanisms.

RelA can interact with the mitochondrial chaperone mortalin, in a manner antagonised by the p53 tumour suppressor, leading to increased O2 consumption and ATP production [23]. Mitochondrial RelA levels also increase during apoptosis driven by ANT1 overexpression and LPS stimulation in cell lines, as well as in rat retina cells after exposure to very bright light [22,26]. Fas-induced apoptosis, on the other hand, reduced mitochondrial RelA and IκBα levels [25]. In all these cases, RelA was required for cell survival, indicating that mitochondrial RelA may play a role in preventing apoptosis. However, IκBα has been reported to be mostly associated with the outer mitochondrial membrane (OMM) with only a small fraction residing inside mitochondria [25,27]. Activation of NF-κB signalling by TNFα or Fas decreases mitochondrial IκBα and plays an important role in preventing apoptosis, independently of its function during NF-κB signalling [20,25]. OMM-associated IκBα mediates the interaction of VDAC and HKII during apoptosis and is necessary for survival [27]. These anti-apoptotic functions of mitochondrial IκBα contribute to tumour survival after doxorubicin treatment [27]. Although a mitochondrial form of p50 has been reported to have DNA binding activity, no functional data are currently available regarding a mitochondrial role for this NF-κB subunit [20,21].

Given these important functions of RelA and IκBα in mitochondrial metabolism and apoptosis, we decided to perform a detailed analysis of both the presence and sub-mitochondrial localisation of all NF-κB subunits. We discovered that only RelA and IκBα were consistently found inside mitochondria, whilst p50 resided in the ER. RelA mitochondrial localisation depended on the OMM translocase TOM40 and the mitochondrial chaperone mortalin. Additionally, we identified hypoxia as a novel stimulus causing a transient increase in mitochondrial RelA and IκBα, independently of the main hypoxia transcription factor subunit HIF1α and calcium signalling. Instead, reactive oxygen species (ROS) production drives RelA, IκBα as well as STAT3 mitochondrial localisation. We also report that localisation of RelA and IκBα to hypoxic mitochondria is dependent on STAT3 signalling, revealing a new and unexpected aspect of crosstalk between these pathways.

Materials and methods

Cell culture

Human Embryonic Kidney 293 (HEK293); breast cancer MCF7 and MDA-MB-231; and osteosarcoma U2OS cell lines were maintained in DMEM supplemented with 10% FBS (Thermo Fisher), 2 mM L-glutamine (SLS) and 50 U Penicillin/Streptomycin (SLS). Prostate cancer PC3 cells were cultured in RPMI with 25 mM HEPES supplemented with 10% FBS (Thermo Fisher) and 2 mM L-glutamine (SLS), whilst primary normal human juvenile dermal fibroblasts (Promocell) were maintained in Fibroblast growth media supplemented with 2% supplement mix (Promocell) and 1% Penicillin/Streptomycin/Fungizone Solution (Promocell).

Treatments

In hypoxia experiments, cells were incubated for the indicated times at 1% O2 in an in vivo 400 hypoxia work station (Ruskin). DMOG (Sigma); Thaspsigargin (Enzo); TPCA1 (Stratech) and STATTIC (Sigma) were dissolved in DMSO. N-acetyl-L-cysteine (SLS) was diluted in water and adjusted to pH 7.5. Cells were treated accordingly at concentrations and times indicated in figure legends.

Mitochondria isolation by differential centrifugation

Cells were homogenised with a 7 ml Dounce homogenizer in homogenisation buffer (0.6 M D-mannitol, 5 mM Tris, pH 7.6; 1 mM EGTA, 0.1% BSA, 1µg/ml Leupeptin, Pepstatin A and Aprotenin and 1 mM PMSF) and centrifuged at 400 × g for 10 min at 4°C to remove cell debris. Cytoplasmic and crude mitochondrial fractions were obtained by centrifugation of the supernatant at 11,000 × g for 10 min at 4°C. Protein concentration was measured by Bradford (BioRad) and the mitochondrial pellets and the cytosolic fraction were treated with 10 ng Proteinase K per µg of protein for 30 min on ice. The Proteinase K treated mitochondria were washed in homogenisation buffer and the concentrations of cytosolic and mitochondrial protein were measured by Bradford assay (Biorad), to allow equal loading for analysis by SDS/PAGE and immunoblotting.

Purification of mitochondria-associated ER

Crude mitochondria were prepared by differential centrifugation and laid on a 15%/17.5%/20% OptiPrep Density Gradient Medium (Sigma). The mitochondria and ER fractions were separated at 100,000 × g with an Optima L-100 XP ultracentrifuge (Beckman Coulter) equipped with a SW40Ti rotor for 2 h at 4°C. The purified ER was harvested from the 20% layer and half was treated with 10 ng Proteinase K per µg of protein for 30 min on ice. Equal amounts of protein were further analysed by immunoblotting.

Mitochondrial sub-fractionation

Crude mitochondria were prepared by differential centrifugation, incubated on a tube rotator with 0.2U DNAse per mg of mitochondria for 15 min at room temperature and sub-fractionated by swelling with 1 mM EDTA/10mM Tris, pH 7.4 on a tube rotator at 4°C. The remaining mitoplast containing only the inner mitochondrial membrane and the enclosed matrix was additionally treated with 5 ng proteinase K/µg of protein for 30 min on ice. Equal amount of protein was further analysed by immunoblotting.

siRNA knock down

U2OS cells were seeded at a density of 400,000 cells/plate in 10 cm2 dishes and transfected in OptiMem (ThermoFisher) with 5 nM siRNA pools or single siRNAs with 5 µl INTERFERin (Polyplus Transfection) transfection reagent the following day. After 48 h, the cells were split into 15 cm2 dishes and transfected again with 7.5 nM siRNA pools with 7.5 µl INTERFERin transfection reagent in OptiMem. The cells were harvested and mitochondria isolated 48 h later by differential centrifugation. Protein levels in the mitochondrial and cytosolic fractions were assessed by Bradford assay (BioRad) and mitochondria were further treated with 10 ng Proteinase K per µg of protein for 30 min on ice. The siRNA used were as follows: Scramble 5′ CAGUCGCGUUUGCGACUGG; RelA siRNA 1 5′ GCUGAUGUGCACCGACAAG; RelA siRNA 2 5′ GCCCUAUCCCUUUACGUCA; IκBα 5′ ACACUCAGCUCAUAAUA; TOM40 siRNA 1 ACUGAACAACUGGUUGG; TOM40 siRNA 2 CCCUCUGUAUGAAAUAG; TOM40 siRNA 3 GAAGAUGACUAUUUCAU; TIM50 siRNA 1 5′ AAUGUUGGCUUCUAACUAA; TIM50 siRNA 2 5′ GGUCUGUACUGUCCAGUAC; Mortalin 5′ CGAGUUGAAGCAGUUAAUA.

Immunoblotting

Protein quantity was measured with Bradford reagent (BioRad) and prepared in SDS loading dye. For detecting HIF1α, a sample of the cells was harvested in 8 M Urea, sonicated and the protein quantified with the BCA assay (ThermoFisher). Between 5 and 10 µg of protein per well was loaded on SDS/PAGE (BioRad), separated and transferred with the Trans Blot Turbo semi dry system (BioRad) to PVDF (Millipore) membranes. The following antibodies were used for the present study at a dilution 1:1000: RelA C-terminal (sc-372, SantaCruz); RelA N-terminal (#4764, Cell Signalling); IκBα (#9242, Cell Signalling); p105/p50 (#3035, Cell Signalling); p100/p52 (#4882, Cell Signalling); RelB (#4954, Cell Signalling); c-Rel (#4727, Cell Signalling); α-tubulin (#2144, Cell Signalling); VDAC (#4866, Cell Signalling); protein disulphide isomerase (PDI) (sc-20132, Santa Cruz); TOM40 (sc-11414, Cell Signalling); AIF (sc-13116, Cell Signalling); TIM50 (SBS0666, Source BioSciense); Mortalin (MA3-028; Thermo Fisher); calnexin (#2433, Cell Signalling); HIF1α (610958, BD Transduction Laboratories); STAT3 (#9139, Cell Signalling).

Immunofluorescence of saponin permeabilised cells

U2OS cells were grown on cover slips and exposed to hypoxia as indicated in figure legends. The cells were permeabilised with Solution A (7.23 mM K2EGTA, 2.77 mM CaK2EGTA, 60 mM MES, 20 mM taurine, 3 mM K2HPO4, 0.5 mM DTT, 81 mM CH3KO3S, pH 7.1) supplemented with 2.88 mg/ml MgATP and 70 µg/ml saponin for 10 min on ice according to [29]. After three washes with Solution A containing MgATP, cells were fixed in 3.7% Formaldehyde for 10 min, permeabilised with 1% Triton-X/0.05% Tween 20 for 15 min, washed in PBS and blocked for 30 min in 10% BSA in PBS/0.05% Tween 20. Slides were stained for 1 h with a 1:200 dilution of RelA sc-372 antibody (Santa Cruz) in 1% BSA/PBS/0.05% Tween20, washed in PBS and stained in the dark with a 1:500 dilution of goat anti-rabbit IgG DyLight 550 (Thermo Fisher) in 1% BSA/PBS/0.05% Tween20 for 30 min. After three more washes with PBS, slides were mounted with ProLong Gold mounting media containing DAPI (Invitrogen) and imaged with a Nikon A1R inverted confocal microscope.

Results

Only RelA from the NF-κB family of transcription factors, as well as IκBα reside inside mitochondria

Several studies have demonstrated that members of the NF-κB signalling pathway can be present within mitochondrial protein extracts, including RelA, p50, IκBα, IKKα, IKKβ and IKKγ, where they are thought to control apoptosis and mitochondrial metabolism [20,21,23,26,27]. However, a full investigation of the mitochondrial presence of all NF-κB subunits has not been undertaken. Moreover, mitochondria are closely associated with the ER [30] and some protocols are not able to distinguish between localisation in different compartments of the mitochondria or potential ER contamination.

To investigate the mitochondrial localisation of NF-κB subunits, crude mitochondria were purified by differential centrifugation of cellular homogenates containing intact organelles from p53 wild-type HEK293 and p53 mutant MDA-MB-231 cells to account for the known inhibitory effects of p53 expression on mitochondrial NF-κB [19,23]. Mitochondria isolated by this method, confirmed by the presence of the mitochondrial outer membrane protein VDAC, were free of cytosolic contamination, as shown by the lack of tubulin (Figure 1A). Examination of all NF-κB subunits in MDA-MB-321 and HEK293 cells corroborated previous findings that RelA, IκBα and p50 are associated with mitochondria [20,21,23,26,27]. Additionally, p105, p100/p52 and c-Rel but not RelB were also associated with the organelle (Figure 1A). However, only RelA and IκBα in both cell lines and p50 in MDA-MB-231 cells were resistant to proteinase K digestion, utilised to remove proteins unshielded from the mitochondrial membranes. This suggested that whilst p50 might only be associated with mitochondria in cells with mutant p53, RelA and IκBα can localise to mitochondria independently of p53 status. (Figure 1A). In HEK293 cell, we also noticed that with the N-terminal RelA antibody we detected a lower mobility band present only in the mitochondria that was also proteinase K resistant. However, RelA siRNA knock down indicated that this band was non-specific (data not shown). This data indicates that even though p105, p100/p52 and c-Rel might exist in proximity to mitochondria, they are unlikely to reside inside the organelle.

RelA and IκBα reside in mitochondria

Figure 1
RelA and IκBα reside in mitochondria

Protein levels from the indicated sub-cellular fractions were analysed by immunoblotting. Mitochondrial and cytosolic extracts were prepared from MDA-MB-231 (A) and HEK293 (A, B) cells by differential centrifugation. Both fractions were treated with 10 ng/µg of protein with proteinase K for 30 min on ice. (C) The ER from MDA-MB-231 cells was separated from crude mitochondrial extracts on a 15%/17.5%/20% OptiPrep gradient. Pure ER was collected in the 20% fraction and treated with 10 ng/µg of protein with proteinase K for 30 min on ice. (D) Mitochondria from HEK293 cells were treated with 0.2 U DNAse per mg of mitochondria for 15 min at room temperature and sub-fractionated by swelling with 10 mM Tris, pH 7.4. The remaining mitoplast containing only the inner mitochondrial membrane and the enclosed matrix was additionally treated with 5 ng/µg of protein with proteinase K from 30 min on ice. (E) RelA and IκBα were knocked down with siRNA in MDA-MB-231 cells and proteinase K-treated mitochondria were prepared as in A. Data are representative of a minimum of two independent experiments.

Figure 1
RelA and IκBα reside in mitochondria

Protein levels from the indicated sub-cellular fractions were analysed by immunoblotting. Mitochondrial and cytosolic extracts were prepared from MDA-MB-231 (A) and HEK293 (A, B) cells by differential centrifugation. Both fractions were treated with 10 ng/µg of protein with proteinase K for 30 min on ice. (C) The ER from MDA-MB-231 cells was separated from crude mitochondrial extracts on a 15%/17.5%/20% OptiPrep gradient. Pure ER was collected in the 20% fraction and treated with 10 ng/µg of protein with proteinase K for 30 min on ice. (D) Mitochondria from HEK293 cells were treated with 0.2 U DNAse per mg of mitochondria for 15 min at room temperature and sub-fractionated by swelling with 10 mM Tris, pH 7.4. The remaining mitoplast containing only the inner mitochondrial membrane and the enclosed matrix was additionally treated with 5 ng/µg of protein with proteinase K from 30 min on ice. (E) RelA and IκBα were knocked down with siRNA in MDA-MB-231 cells and proteinase K-treated mitochondria were prepared as in A. Data are representative of a minimum of two independent experiments.

Further analysis demonstrated that mitochondria isolated through differential centrifugation also contained a proteinase K resistant fraction of the ER, confirmed by the presence of the ER lumen chaperone PDI (Figure 1B). Although this observation is not unusual, given the physical and functional interactions between these organelles [30], it was essential to discount the possibility of ER localisation of RelA, IκBα or p50. Crude ER/mitochondria preparations from MDA-MB-231 cells were, therefore, further separated on a Percol gradient to obtain a purified ER fraction. Proteinase K treatment of this fraction revealed that although RelA and IκBα were degraded, p50 remained intact in the ER, suggesting that its apparent localisation in mitochondrial extracts in MDA-MB-231 cells (Figure 1A) might originate from contaminating ER (Figure 1C). We therefore only focused on RelA and IκBα in subsequent experiments.

Next, mitochondria were subjected to further fractionation by swelling and removal of the OMM to obtain the mitoplast, the matrix enveloped by the inner mitochondrial membrane (IMM) that contains the mitochondrial genome. Interestingly, RelA localised partially to the proteinase K-treated mitoplast whilst IκBα did not (Figure 1D).

To confirm antibody specificity, we performed RelA or IκBα siRNA knockdown in highly transfectable MDA-MB-231 cells and observed reduced signals for both proteins in mitochondria (Figure 1E). RelA knockdown also reduced the levels of cytosolic IκBα, although this may reflect a reduction in IκBα transcription, which is under the control of RelA [31]. The loss of mitochondrial RelA following IκBα siRNA knockdown was unexpected and could be due to IκBα acting as a chaperone to RelA.

TOM40 and mortalin are required for RelA mitochondrial localisation

Most mitochondrial proteins are nuclear encoded and need to be imported into mitochondria post-translation. The classical pathway for mitochondrial import is through the translocase of the outer membrane (TOM) followed by the translocase of the inner membrane (TIM) [32]. These are large multiprotein complexes forming pores through the mitochondrial membranes to deliver proteins into the intermembrane space and matrix by using ATP and the mitochondrial membrane potential.

To identify which translocase is involved in RelA localisation into mitochondria we used siRNA to knockdown subunits of both translocases in highly transfectable U2OS cells and obtained proteinase K treated-mitochondria by differential centrifugation. RelA mitochondrial import depended on TOM40, a component of the TOM complex that forms a barrel traversing the outer mitochondrial membrane (Figure 2A). However, TIM50, one of three integral membrane proteins of the TIM translocase, was not involved in intrinsic RelA mitochondrial localisation (Figure 2B), although this experiment did not examine localisation to the mitoplast.

TOM40 and mortalin are required for the mitochondrial import of RelA

Figure 2
TOM40 and mortalin are required for the mitochondrial import of RelA

Mitochondrial and cytosolic extracts were prepared by differential centrifugation from U2OS cells, where TOM40 (A), TIM50 (B) or mortalin (C) were knocked down with siRNA. The mitochondrial fractions were treated with 10 ng/µg of protein with proteinase K for 30 min on ice. Protein levels in each fraction were analysed by immunoblotting as indicated. Data are representative of a minimum of two independent experiments.

Figure 2
TOM40 and mortalin are required for the mitochondrial import of RelA

Mitochondrial and cytosolic extracts were prepared by differential centrifugation from U2OS cells, where TOM40 (A), TIM50 (B) or mortalin (C) were knocked down with siRNA. The mitochondrial fractions were treated with 10 ng/µg of protein with proteinase K for 30 min on ice. Protein levels in each fraction were analysed by immunoblotting as indicated. Data are representative of a minimum of two independent experiments.

Our previous findings identified the matrix protein mortalin as a RelA interactor, crucial for its mitochondrial import [23]. Indeed, mortalin knockdown reduced the mitochondrial levels of RelA, confirming that it is needed for RelA mitochondrial localisation (Figure 2C).

Hypoxia triggers rapid and transient mitochondrial translocation of RelA and IκBα

We were curious about the functional significance of RelA and IκBα localisation to mitochondria, specifically in the context of cellular signalling known to regulate their activity. A number of nuclear transcription factors have been found to translocate to mitochondria during hypoxia [33–35]. Therefore, we asked if RelA and IκBα could also do so by exposing cells to 1% O2 and preparing proteinase K-treated mitochondria. Indeed, we observed that both RelA and IκBα translocated to mitochondria within minutes of cells being subjected to hypoxia, with levels decreasing after 1 h (Figure 3A). This was further confirmed by immunofluorescence (Figure 3B,C). Due to the high cytosolic presence of RelA, normoxic and hypoxic U2OS cells were first permeabilised with saponin and the cytosol was washed out, leaving only organelle associated RelA. Analysis of confocal images revealed that RelA colocalised with mitochondria significantly more during hypoxia (Figure 3B,C). It is, therefore, likely that RelA and IκBα are implicated in the early stages of hypoxia, rather than during later stages of adaptation via transcription.

Hypoxia induces a transient RelA and IκBα translocation into mitochondria

Figure 3
Hypoxia induces a transient RelA and IκBα translocation into mitochondria

(A) Cytosolic and mitochondrial extracts were prepared by differential centrifugation from HEK293 and MCF7 cells cultured for the indicated times at 1% O2. Protein levels in each fraction were assessed by immunoblotting as indicated. Data are representative of a minimum of two independent experiments. (B) U2OS cells were grown on cover slips, mitochondria were stained with MitoTracker DeepRed FM (Invitrogen), exposed to 1% O2 for 30 min, cytosolic RelA was removed by permeabilisation with 70 µg/ml saponin on ice under hypoxia and fixed with formaldehyde. Organelle-associated RelA was stained with sc-372 primary antibody (Santa Cruz) and goat anti-rabbit IgG DyLight 550 (Thermo Fisher). Confocal Z stack images were obtained with a Nikon A1R inverted microscope. (C) A total of eight normoxic and eight hypoxic cells from three images each were analysed with the Huygens Colocalization Analyser [64]. Regions of interest that include the mitochondria but exclude the nucleus in individual cells were measured and the Manders’ Overlap Coefficient was calculated for each cell. The values depicted in blue on the graph correspond to the cells shown in (B). The error bars represent standard deviation and statistical analysis was performed in Prism Software with a two-tailed, unpaired t-test.

Figure 3
Hypoxia induces a transient RelA and IκBα translocation into mitochondria

(A) Cytosolic and mitochondrial extracts were prepared by differential centrifugation from HEK293 and MCF7 cells cultured for the indicated times at 1% O2. Protein levels in each fraction were assessed by immunoblotting as indicated. Data are representative of a minimum of two independent experiments. (B) U2OS cells were grown on cover slips, mitochondria were stained with MitoTracker DeepRed FM (Invitrogen), exposed to 1% O2 for 30 min, cytosolic RelA was removed by permeabilisation with 70 µg/ml saponin on ice under hypoxia and fixed with formaldehyde. Organelle-associated RelA was stained with sc-372 primary antibody (Santa Cruz) and goat anti-rabbit IgG DyLight 550 (Thermo Fisher). Confocal Z stack images were obtained with a Nikon A1R inverted microscope. (C) A total of eight normoxic and eight hypoxic cells from three images each were analysed with the Huygens Colocalization Analyser [64]. Regions of interest that include the mitochondria but exclude the nucleus in individual cells were measured and the Manders’ Overlap Coefficient was calculated for each cell. The values depicted in blue on the graph correspond to the cells shown in (B). The error bars represent standard deviation and statistical analysis was performed in Prism Software with a two-tailed, unpaired t-test.

Elevated mitochondrial RelA and IκBα levels during hypoxia are ROS dependent

Hypoxia activates NF-κB via IKK, independently of signalling through the oxygen sensing hypoxia-inducible transcription factor HIF1α, although in hypoxia activation does not involve degradation of IκBα [36,37]. However, lack of oxygen in cells and tissues leads to several additional rapid physiological changes, occurring within minutes of exposure to hypoxia, such as a burst of mitochondrial ROS and superoxide levels [38,39], an increase in intracellular Ca2+ levels [40,41] and accumulation of HIF1α [42]. ROS and superoxide generation during hypoxia mainly originates from the Q cycle in complex III of the mitochondrial electron transport chain [43,44] and is accumulated in the first 30 min after which levels return to basal level [39,45].

To determine if any of these events are involved in mitochondrial RelA and IκBα localisation during hypoxia, we first treated MDA-MB-231 cells with the hypoxia mimetic DMOG, which inhibits the PHD enzymes responsible for HIF1α hydroxylation and proteolytic degradation, leading to accumulation of HIF1α without lowering O2 levels.

DMOG treatment did not alter mitochondrial RelA or IκBα levels (Figure 4A). This finding was reinforced by using a chemical inhibitor of HIF1α translation, thapsigargin that blocks the production of HIF1α protein by preventing the association of the RNA binding protein YB1 with the 5′UTR of HIF1α mRNA [46]. Thapsigargin is also known as a sarco/endoplasmic reticulum Ca2+−ATPase (SERCA) inhibitor that depletes the ER calcium stores and results in accumulation of intracellular Ca2+, another event associated with early hypoxia. Cells treated with thapsigargin showed no changes to RelA and IκBα mitochondrial translocation either with or without hypoxia exposure (Figure 4B). Taken together these findings suggest that HIF1α hydroxylation and accumulation, as well as Ca2+ release do not appear to trigger RelA and IκBα mitochondrial translocation (Figure 4A,B). Instead, cells treated with the antioxidant N-acetyl-L-cysteine (NAC) prevented the mitochondrial translocation of RelA and IκBα during hypoxia, suggesting mitochondrial ROS production is required for mitochondrial RelA and IκBα accumulation (Figure 4C).

Mitochondrial translocation of RelA and IκBα during hypoxia is dependent on ROS

Figure 4
Mitochondrial translocation of RelA and IκBα during hypoxia is dependent on ROS

(A) Cytosolic and mitochondrial extracts were prepared by differential centrifugation from MDA-MB-231 cells treated with 1 mM of the hypoxia mimetic DMOG for 1 and 4 h (B). MCF7 cells were treated with 5 µM Thapsigargin (C) or 10 mM NAC for 1h (D) and then exposed to 1% O2 for 30 min before mitochondria and cytosolic fractions were isolated. All mitochondrial fractions were treated with 10 ng/µg of protein with proteinase K for 30 min on ice. Protein levels in each fraction were assessed by immunoblotting as indicated. Data are representative of a minimum of two independent experiments.

Figure 4
Mitochondrial translocation of RelA and IκBα during hypoxia is dependent on ROS

(A) Cytosolic and mitochondrial extracts were prepared by differential centrifugation from MDA-MB-231 cells treated with 1 mM of the hypoxia mimetic DMOG for 1 and 4 h (B). MCF7 cells were treated with 5 µM Thapsigargin (C) or 10 mM NAC for 1h (D) and then exposed to 1% O2 for 30 min before mitochondria and cytosolic fractions were isolated. All mitochondrial fractions were treated with 10 ng/µg of protein with proteinase K for 30 min on ice. Protein levels in each fraction were assessed by immunoblotting as indicated. Data are representative of a minimum of two independent experiments.

STAT3 signalling is required for RelA and IκBα mitochondrial accumulation during hypoxia

Hypoxia can activate NF-κB within 5 min of exposure via IKKβ [36]. These kinetics are strikingly similar to our observation of RelA and IκBα mitochondrial translocation within 5 min of hypoxia (Figure 3A). To establish if IKK signalling was needed for mitochondrial translocation of RelA and IκBα, we utilised the ATP competitive IKKβ inhibitor TPCA1. TPCA1 is also known to inhibit STAT3, a nuclear transcription factor that translocates to mitochondria during hypoxia, by binding to its SH2 dimerisation domain and preventing phosphorylation of adjacent sites required for activity [33,47]. To address this off target effect, we obtained proteinase K-treated mitochondria from the STAT3 negative cell line PC3 [48]. TPCA1 reduced mitochondrial RelA and IκBα levels in PC3 cells during hypoxia, indicating that active NF-κB signalling is required for their mitochondrial accumulation (Figure 5A). We also observed an increase of RelA and IκBα in normoxia following TPCA1 treatment, which could be due to off target effects.

Inhibition of NF-κB or STAT3 signalling impairs hypoxia dependent RelA and IκBα mitochondrial translocation

Figure 5
Inhibition of NF-κB or STAT3 signalling impairs hypoxia dependent RelA and IκBα mitochondrial translocation

PC3 cells were treated with 10 µM TPCA1 (A) or MCF7 cells were treated with 20 µM STATTIC (B) then exposed to 1% O2 for 30 min. Cytosolic and mitochondrial extracts were prepared by differential centrifugation and mitochondrial fractions were incubated with 10 ng/µg of protein with proteinase K for 30 min on ice. (C) Hypoxia- and NAC-treated extracts from Figure 3D were analysed by immunoblotting for STAT3 levels. Data are representative of a minimum of two independent experiments.

Figure 5
Inhibition of NF-κB or STAT3 signalling impairs hypoxia dependent RelA and IκBα mitochondrial translocation

PC3 cells were treated with 10 µM TPCA1 (A) or MCF7 cells were treated with 20 µM STATTIC (B) then exposed to 1% O2 for 30 min. Cytosolic and mitochondrial extracts were prepared by differential centrifugation and mitochondrial fractions were incubated with 10 ng/µg of protein with proteinase K for 30 min on ice. (C) Hypoxia- and NAC-treated extracts from Figure 3D were analysed by immunoblotting for STAT3 levels. Data are representative of a minimum of two independent experiments.

To further delineate the pathway required for RelA and IκBα mitochondrial accumulation, we utilised STATTIC, an irreversible inhibitor of STAT3. Similarly to TPCA1, STATTIC also inhibits the SH2 dimerisation domain and adjacent phosphorylation sites of STAT3, resulting in lack of DNA binding and loss of Tyr705 and Ser727 phosphorylation [49,50]. Interestingly, STAT3 positive MCF7 cells treated with this inhibitor accumulated less STAT3, RelA and IκBα into mitochondria during hypoxia, suggesting that active STAT3 can also regulate the mitochondrial association of these factors (Figure 5B). Moreover, NAC also reduced mitochondrial STAT3 levels in cells undergoing hypoxia, implicating ROS in mitochondrial STAT3 signalling (Figure 5C). This is consistent with the role of mitochondria targeted STAT3 in decreasing ROS production by the electron transport chain of ischemic mouse hearts [33]. It is therefore likely that hypoxia-associated ROS production acts as a signal to rapidly recruit STAT3, RelA and IκBα to mitochondria.

Discussion

In the present study, we established that of the NF-κB subunits, only RelA together with its inhibitor IκBα, are consistently found inside the mitochondria of unstimulated cancer cells (Figure 1A,C). Moreover, we investigated the mitochondrial import route for RelA (Figure 2). As described previously, we confirmed that the mitochondrial chaperon mortalin interacts with RelA and is required for RelA mitochondrial localisation (Figure 2C) [23]. Mortalin aids the import of a subset of mitochondrial proteins by binding to them in the cytosol and facilitating their docking to the OMM translocase [51,52]. The OMM component TOM40 was also required for mitochondrial RelA localisation (Figures 1D and 2A). A proportion of RelA was also found in the mitoplast (Figure 1D). This biochemical evidence is consistent with previous ChIP data demonstrating that RelA can alter mitochondrial metabolism by associating with mtDNA indirectly [23]. In contrast, IκBα was not found in the mitoplast, suggesting it functions differently to RelA, perhaps in preventing apoptosis as previously shown [27].

Intriguingly, we identified hypoxia as a novel stimulus resulting in rapid and transient accumulation of RelA and IκBα into mitochondria, independently of HIF1α accumulation and Ca2+ release (Figures 3 and 4). STAT3 also accumulated rapidly in mitochondria following hypoxia (Figure 5B,C). STAT3 mitochondrial import is well defined. Similarly to RelA, STAT3 also uses a chaperone for import as well as for integration into complex I within the IMM [53]. Furthermore, Ser727 phosphorylation and K87 acetylation of STAT3 are crucial for mitochondrial import [53,54].

Inhibition of NF-κB and STAT3 signalling, with TPCA1 and STATTIC, respectively, reduced mitochondrial RelA and IκBα during hypoxia (Figure 5). STATTIC and TPCA1 can both prevent Ser727 phosphorylation of STAT3 [47,49,50], thereby, inhibiting STAT3 mitochondrial localisation simply by reducing mitochondrial import. STATTIC has also been found to reduce NF-κB activation and DNA binding, presumably due to crosstalk with STAT3 signalling [50,55]. A direct inhibitory effect of STATTIC on NF-κB, however, cannot be excluded.

STATTIC treatment also leads to increased mitochondrial ROS production and opening of the mitochondrial permeability transition pore (MPTP), a conductance channel at the IMM that leads to mitochondrial swelling, rupture of the OMM and apoptosis [56,57]. We therefore can also not rule out that reduction of RelA and IκBα mitochondrial levels after STATTIC treatment might be due to loss of the integrity of the mitochondrial membranes and consequent leakage of mitochondrial proteins.

Mitochondrial STAT3 regulates mitochondrial complex I and complex II activity [58,59]; controls pyruvate to acetyl-CoA conversion, ATP production, fatty acid synthesis and the mitochondrial potential following insulin stimulation [54]. It also suppresses mitochondrial ROS production, preventing MPTP opening and apoptosis [56–58,60,61]. It is therefore likely that during hypoxia STAT3 might play a similar role in suppressing ROS production and apoptosis (Figure 5C).

Mitochondrial ROS generated during hypoxia can activate the redox sensing tyrosine kinase c-SRC which in turn can stimulate both NF-κB and STAT3 through tyrosine phosphorylation on IκBα and STAT3 [37,62]. Interestingly, the kinetics of rapid ROS accumulation observed in the literature [39,45] mirror the kinetics of RelA and IκBα accumulation following exposure to hypoxia (Figure 3A). Consistent with ROS being implicated in activation of these pathways, we observed that the antioxidant NAC reduced mitochondrial RelA, IκBα and STAT3 during hypoxia (Figures 4C and 5C).

Similar to STAT3, IκBα has also been implicated in preventing apoptosis [27]. Inhibition of NF-kB signalling following hypoxia in ex vivo cultured rat ventricular myocytes leads to mitochondrial potential loss, MPTP opening and caspase 9 activation, suggestion that the anti-apoptotic role of NF-κB is mediated through the mitochondria [63]. Taken together our data and the available literature point to a scenario in which mitochondrial RelA, IκBα and STAT3 cooperatively participate in preventing apoptosis during the early stages of hypoxia.

Acknowledgments

We would like to thank Dr. Francesco Bruni and Dr. Nicole Mai as well as all other members of the Lightowlers group at the Newcastle University Wellcome Trust Centre for Mitochondrial Research for sharing mitochondria extraction protocols and many useful discussions. We would also like to acknowledge Dr. Rolando Berlinguer Palmini from the BioImaging Unit, Newcastle University and Dr. Adrian Yemm from the Perkins laboratory for help with microscopy analysis.

Funding

This work was supported by the Cancer Research U.K. [grant number C1443/A22095].

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Author Contribution

I.G.I. performed experimental work; I.G.I. and N.D.P. contributed to experimental design and manuscript preparation.

Abbreviations

     
  • ANT1

    Adenine Nucelotide Transporter 1

  •  
  • ChlP

    chromatin immunoprecipitation

  •  
  • c-Rel

    REL Proto-Oncogene, NF-κB subunit

  •  
  • c-SRC

    SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase

  •  
  • DMOG

    dimethyloxaloylglycine

  •  
  • DTT

    Dithiothreitol

  •  
  • ER

    endoplasmic reticulum

  •  
  • HEK293

    human embryonic kidney 293

  •  
  • HEPES

    (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid

  •  
  • HIF1α

    Hypoxia Inducilbe Factor 1α

  •  
  • HKII

    Hexokinase 2

  •  
  • IκB

    inhibitor of NF-κB

  •  
  • IKK

    IκB kinase

  •  
  • IMM

    inner mitochondrial membrane

  •  
  • LPS

    Lipopolysaccharide

  •  
  • MES

    2-(N-morpholino)ethanesulfonic acid

  •  
  • MPTP

    mitochondrial permeability transition pore

  •  
  • mtDNA

    mitochondrial DNA

  •  
  • NAC

    N-acetyl-L-cysteine

  •  
  • NEMO

    NF-κB Essential Modulator

  •  
  • NF-κB

    nuclear factor-κB

  •  
  • OMM

    outer mitochondrial membrane

  •  
  • PMSF

    Phenylmethylsulfonyl fluoride

  •  
  • p65

    RELA Proto-Oncogene, NF-κB subunit

  •  
  • RelA

    RELA Proto-Oncogene, NF-κB subunit

  •  
  • RelB

    RELA Proto-Oncogene, NF-κB subunit

  •  
  • ROS

    reactive oxygen species

  •  
  • RPMI

    Roswell Park Memorial Institute (RPMI) 1640 medium

  •  
  • SLS

    Scientific Laboratory Supplies Ltd

  •  
  • STATTIC

    Selective STAT3 inhibitor, 6-Nitrobenzo[b]thiophene 1,1 dioxide

  •  
  • STAT3

    Signal Transducer and Activator of Transcription 3

  •  
  • TIM

    translocase of the outer membrane

  •  
  • TOM

    translocase of the outer membrane

  •  
  • TPCA1

    2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide

  •  
  • VDAC

    Voltage Dependent Anion Channel

References

References
1.
Zhao
M.
et al. .
(
2018
)
Transcriptional outcomes and kinetic patterning of gene expression in response to NF-κB activation
.
PLoS Biol.
16
,
e2006347
2.
Hoesel
B.
and
Schmid
J.A.
(
2013
)
The complexity of NF-κB signaling in inflammation and cancer
.
Mol. Cancer
12
,
86
3.
Xia
Y.
,
Shen
S.
and
Verma
I.M.
(
2014
)
NF-κB, an active player in human cancers
.
Cancer Immunol. Res.
2
,
823
830
4.
Phelps
C.B.
et al. .
(
2000
)
Mechanism of κB DNA binding by Rel/NF-κB dimers
.
J. Biol. Chem.
275
,
24392
24399
5.
Berkowitz
B.
et al. .
(
2002
)
The x-ray crystal structure of the NF-κB p50.p65 heterodimer bound to the interferon beta -κB site
.
J. Biol. Chem.
277
,
24694
24700
6.
Schmitz
M.L.
,
dos Santos Silva
M.A.
and
Baeuerle
P.A.
(
1995
)
Transactivation domain 2 (TA2) of p65 NF-κB. Similarity to TA1 and phorbol ester-stimulated activity and phosphorylation in intact cells
.
J. Biol. Chem.
270
,
15576
15584
7.
Dobrzanski
P.
,
Ryseck
R.P.
and
Bravo
R.
(
1993
)
Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif
.
Mol. Cell. Biol.
13
,
1572
1582
8.
Huxford
T.
et al. .
(
1998
)
The crystal structure of the IκBα/NF-κB complex reveals mechanisms of NF-κB inactivation
.
Cell
95
,
759
770
9.
Hatada
E.N.
et al. .
(
1992
)
The ankyrin repeat domains of the NF-κB precursor p105 and the protooncogene bcl-3 act as specific inhibitors of NF-κB DNA binding
.
Proc. Natl. Acad. Sci. U.S.A.
89
,
2489
2493
10.
Ganchi
P.A.
et al. .
(
1992
)
IκB/MAD-3 masks the nuclear localization signal of NF-κB p65 and requires the transactivation domain to inhibit NF-κB p65 DNA binding
.
Mol. Biol. Cell
3
,
1339
1352
11.
Betts
J.C.
and
Nabel
G.J.
(
1996
)
Differential regulation of NF-κB2(p100) processing and control by amino-terminal sequences
.
Mol. Cell. Biol.
16
,
6363
6371
12.
Moorthy
A.K.
et al. .
(
2006
)
The 20S proteasome processes NF-κB1 p105 into p50 in a translation-independent manner
.
EMBO J.
25
,
1945
1956
13.
Israel
A.
(
2010
)
The IKK complex, a central regulator of NF-κB activation
.
Cold Spring Harb. Perspect. Biol.
2
,
a000158
14.
Perkins
N.D.
(
2012
)
The diverse and complex roles of NF-κB subunits in cancer
.
Nat. Rev. Cancer
12
,
121
132
15.
Perkins
N.D.
(
2018
)
More than just an IκB kinase: the IKK complex coordinates mRNA stability and transcription
.
EMBO J.
37
,
e101084
16.
Wallace
D.C.
(
1999
)
Mitochondrial diseases in man and mouse
.
Science
283
,
1482
1488
17.
Suomalainen
A.
and
Battersby
B.J.
(
2018
)
Mitochondrial diseases: the contribution of organelle stress responses to pathology
.
Nat. Rev. Mol. Cell Biol.
19
,
77
92
18.
Laforge
M.
et al. .
(
2016
)
NF-κB pathway controls mitochondrial dynamics
.
Cell Death Differ.
23
,
89
98
19.
Mauro
C.
et al. .
(
2011
)
NF-κB controls energy homeostasis and metabolic adaptation by upregulating mitochondrial respiration
.
Nat. Cell Biol.
13
,
1272
1279
20.
Cogswell
P.C.
et al. .
(
2003
)
NF-κB and IκB α are found in the mitochondria. Evidence for regulation of mitochondrial gene expression by NF-κB
.
J. Biol. Chem.
278
,
2963
2968
21.
Guseva
N.V.
et al. .
(
2004
)
Tumor necrosis factor-related apoptosis-inducing ligand-mediated activation of mitochondria-associated nuclear factor-κB in prostatic carcinoma cell lines
.
Mol. Cancer Res.
2
,
574
584
22.
Tomita
H.
et al. .
(
2016
)
Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina
.
Biochem. Biophys. Res. Commun.
473
,
1013
1018
23.
Johnson
R.F.
,
Witzel
II
and
Perkins
N.D.
(
2011
)
p53-dependent regulation of mitochondrial energy production by the RelA subunit of NF-κB
.
Cancer Res.
71
,
5588
5597
24.
Song
Y.
et al. .
(
2018
)
NF-κB in mitochondria regulates PC12 cell apoptosis following lipopolysaccharide-induced injury
.
Biomed. Biotechnol.
19
,
425
435
25.
Bottero
V.
et al. .
(
2001
)
IκB-α, the NF-κB inhibitory subunit, interacts with ANT, the mitochondrial ATP/ADP translocator
.
J. Biol. Chem.
276
,
21317
21324
26.
Zamora
M.
et al. .
(
2004
)
Recruitment of NF-κB into mitochondria is involved in adenine nucleotide translocase 1 (ANT1)-induced apoptosis
.
J. Biol. Chem.
279
,
38415
38423
27.
Pazarentzos
E.
et al. .
(
2014
)
IκBα inhibits apoptosis at the outer mitochondrial membrane independently of NF-κB retention
.
EMBO J.
33
,
2814
2828
28.
Marinov
G.K.
et al. .
(
2014
)
Evidence for site-specific occupancy of the mitochondrial genome by nuclear transcription factors
.
PLoS ONE
9
,
e84713
29.
Safiulina
D.
et al. .
(
2004
)
Method for in situ detection of the mitochondrial function in neurons
.
J. Neurosci. Methods
137
,
87
95
30.
Szymanski
J.
et al. .
(
2017
)
Interaction of mitochondria with the endoplasmic reticulum and plasma membrane in calcium homeostasis, lipid trafficking and mitochondrial structure
.
Int. J. Mol. Sci.
18
,
pii: E1576
31.
Scott
M.L.
et al. .
(
1993
)
The p65 subunit of NF-κB regulates IκB by two distinct mechanisms
.
Genes Dev.
7
,
1266
1276
32.
Rehling
P.
,
Brandner
K.
and
Pfanner
N.
(
2004
)
Mitochondrial import and the twin-pore translocase
.
Nat. Rev. Mol. Cell Biol.
5
,
519
530
33.
Szczepanek
K.
et al. .
(
2011
)
Mitochondrial-targeted signal transducer and activator of transcription 3 (STAT3) protects against ischemia-induced changes in the electron transport chain and the generation of reactive oxygen species
.
J. Biol. Chem.
286
,
29610
29620
34.
Steven
A.
et al. .
(
2016
)
Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization
.
Oncotarget
7
,
52061
52084
35.
Sansome
C.
et al. .
(
2001
)
Hypoxia death stimulus induces translocation of p53 protein to mitochondria. Detection by immunofluorescence on whole cells
.
FEBS Lett.
488
,
110
115
36.
Culver
C.
et al. .
(
2010
)
Mechanism of hypoxia-induced NF-κB
.
Mol. Cell. Biol.
30
,
4901
4921
37.
Lluis
J.M.
et al. .
(
2007
)
Dual role of mitochondrial reactive oxygen species in hypoxia signaling: activation of nuclear factor-{κ}B via c-SRC and oxidant-dependent cell death
.
Cancer Res.
67
,
7368
7377
38.
Angelos
M.G.
et al. .
(
2006
)
Hypoxic reperfusion of the ischemic heart and oxygen radical generation
.
Am. J. Physiol. Heart Circ. Physiol.
290
,
H341
H347
39.
Hernansanz-Agustin
P.
et al. .
(
2014
)
Acute hypoxia produces a superoxide burst in cells
.
Free Radic. Biol. Med.
71
,
146
156
40.
Gusarova
G.A.
et al. .
(
2011
)
Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation
.
Mol. Cell. Biol.
31
,
3546
3556
41.
Nayler
W.G.
,
Poole-Wilson
P.A.
and
Williams
A.
(
1979
)
Hypoxia and calcium
.
J. Mol. Cell Cardiol.
11
,
683
706
42.
Jewell
U.R.
et al. .
(
2001
)
Induction of HIF-1α in response to hypoxia is instantaneous
.
FASEB J.
15
,
1312
1314
43.
Hamanaka
R.B.
and
Chandel
N.S.
(
2009
)
Mitochondrial reactive oxygen species regulate hypoxic signaling
.
Curr. Opin. Cell Biol.
21
,
894
899
44.
Poyton
R.O.
,
Ball
K.A.
and
Castello
P.R.
(
2009
)
Mitochondrial generation of free radicals and hypoxic signaling
.
Trends Endocrinol. Metab.
20
,
332
340
45.
Hernansanz-Agustín
P.
et al. .
(
2018
)
Mitochondrial Na+ import controls oxidative phosphorylation and hypoxic redox signalling
.
bioRxiv
46.
Ivanova
I.G.
et al. .
(
2018
)
PERK/eIF2α signaling inhibits HIF-induced gene expression during the unfolded protein response via YB1-dependent regulation of HIF1α translation
.
Nucleic Acids Res.
46
,
3878
3890
47.
Nan
J.
et al. .
(
2014
)
TPCA-1 is a direct dual inhibitor of STAT3 and NF-κB and regresses mutant EGFR-associated human non-small cell lung cancers
.
Mol. Cancer Ther.
13
,
617
629
48.
Seim
I.
et al. .
(
2017
)
Whole-genome sequence of the metastatic PC3 and LNCaP human prostate cancer cell lines
.
G3 (Bethesda)
7
,
1731
1741
49.
Schust
J.
et al. .
(
2006
)
Stattic: a small-molecule inhibitor of STAT3 activation and dimerization
.
Chem. Biol.
13
,
1235
1242
50.
Li
C.H.
et al. .
(
2018
)
Stattic inhibits RANKL-mediated osteoclastogenesis by suppressing activation of STAT3 and NF-κB pathways
.
Int. Immunopharmacol.
58
,
136
144
51.
Schneider
H.C.
et al. .
(
1994
)
Mitochondrial Hsp70/MIM44 complex facilitates protein import
.
Nature
371
,
768
774
52.
Young
J.C.
,
Hoogenraad
N.J.
and
Hartl
F.U.
(
2003
)
Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70
.
Cell
112
,
41
50
53.
Tammineni
P.
et al. .
(
2013
)
The import of the transcription factor STAT3 into mitochondria depends on GRIM-19, a component of the electron transport chain
.
J. Biol. Chem.
288
,
4723
4732
54.
Xu
Y.S.
et al. .
(
2016
)
STAT3 undergoes acetylation-dependent mitochondrial translocation to regulate pyruvate metabolism
.
Sci. Rep.
6
,
39517
55.
Fan
P.
et al. .
(
2018
)
Modulation of nuclear factor-κB activation by the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in breast cancer cells
.
Cell Death Discov.
4
,
15
56.
Lachance
C.
,
Goupil
S.
,
Leclerc
P.
and
Stattic
V
(
2013
)
a STAT3 inhibitor, affects human spermatozoa through regulation of mitochondrial activity
.
J. Cell. Physiol.
228
,
704
713
57.
Boengler
K.
et al. .
(
2013
)
The STAT3 inhibitor stattic impairs cardiomyocyte mitochondrial function through increased reactive oxygen species formation
.
Curr. Pharm. Des.
19
,
6890
6895
58.
Boengler
K.
et al. .
(
2010
)
Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion
.
Basic Res. Cardiol.
105
,
771
785
59.
Wegrzyn
J.
et al. .
(
2009
)
Function of mitochondrial Stat3 in cellular respiration
.
Science
323
,
793
797
60.
Meier
J.A.
et al. .
(
2017
)
Stress-induced dynamic regulation of mitochondrial STAT3 and its association with cyclophilin D reduce mitochondrial ROS production
.
10
,
pii: eaag2588
61.
Cheng
X.
,
Peuckert
C.
and
Wolfl
S.
(
2017
)
Essential role of mitochondrial Stat3 in p38(MAPK) mediated apoptosis under oxidative stress
.
Sci. Rep.
7
,
15388
62.
Sato
H.
et al. .
(
2005
)
Mitochondrial reactive oxygen species and c-Src play a critical role in hypoxic response in vascular smooth muscle cells
.
Cardiovasc. Res.
67
,
714
722
63.
Regula
K.M.
,
Baetz
D.
and
Kirshenbaum
L.A.
(
2004
)
Nuclear factor-κB represses hypoxia-induced mitochondrial defects and cell death of ventricular myocytes
.
Circulation
110
,
3795
3802
64.
Dunn
K.W.
,
Kamocka
M.M.
and
McDonald
J.H.
(
2011
)
A practical guide to evaluating colocalization in biological microscopy
.
Am. J. Physiol. Cell Physiol.
300
,
C723
C742
This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).