Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70

Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional prosurvival protein that binds to several intracellular targets and promotes cell survival. HSP70 and Raf-1 are important targets of Bag-1; however, the protective function of Bag-1 in nucleus pulposus (NP) cells remains unclear. In this study, we determined the effects of Bag-1 on NP cells under oxidative stress induced by treatment with hydrogen peroxide (H2O2). We found that Bag-1 was bound to HSP70, but Bag-1–Raf1 binding did not occur in NP cells. Bag-1 overexpression in NP cells enhanced cell viability and mitochondrial function and significantly suppressed p38/MAPKs phosphorylation during oxidative stress, although NP cells treated with a Bag-1 C-terminal inhibitor, which is the binding site of HSP70 and Raf-1, decreased cell viability and mitochondrial function during oxidative stress. Furthermore, the phosphorylation of the ERK/MAPKs was significantly increased in Bag-1 C-terminal inhibitor-treated NP cells without H2O2 treatment but did not change with H2O2 exposure. The phosphorylation of Raf-1 was not influenced by Bag-1 overexpression or Bag-1 C-terminal binding site inhibition. Overall, the results suggest that Bag-1 preferentially interacts with HSP70, rather than Raf-1, to protect NP cells against oxidative stress.


Introduction
The intervertebral disc (IVD) is an organ that exists between the vertebrae. It is composed of an outer fibrocartilaginous annulus fibrosus (AF) that surrounds a gel-like nucleus pulposus (NP). The IVD is an avascular tissue that exchanges nutrients and metabolites primarily by diffusion to and from microvessels in the superior and inferior cartilaginous end plate of the vertebrae and outer AF [1][2][3]. The NP consists of a small number of NP cells scattered in the extracellular matrix, which absorb shock and maintain spinal mobility. NP cells have adapted to survive in this unique, hypoxic environment [4]. As a result of aging, mechanical stress, genetics, and many other factors, the number of functional NP cells decreases through apoptosis and cellular senescence. This is accompanied by a phenotypic shift toward catabolism and results in gradual IVD degeneration. IVD degeneration is clinically related to disc herniation, spinal canal stenosis, spinal deformities, and chronic low back pain, which has a profound effect on patient quality of life and causes a considerable socioeconomic burden [1,2]. Recent studies have shown that oxidative stress resulting from excessive reactive oxygen species (ROS) promotes apoptosis, necrosis, and senescence of NP cells in the disc microenvironment [5][6][7][8][9]. Excessive ROS can induce mitochondrial dysfunction, including mitochondrial membrane potential collapse, ultrastructure disintegration, and ATP depletion. This results in additional ROS accumulation as well as DNA damage, cell senescence, and disruption of NP homeostasis [10][11][12].

Isolation of NP Cells, Hypoxic Culture Conditions, and Cell Treatments
Rat NP cells were isolated from male Sprague-Dawley rats (11 weeks old) using a modified method described by Risbud et al. [30]. Briefly, IVDs from the lumbar and coccygeal regions were dissected from rats under deep aesthesia using aseptic conditions. The gel-like NP tissue was separated from the AF and the NP tissue was minced by pipetting. The isolated cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Nakarai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) antibiotics and cultured in a Hypoxia Chamber (MIC-101; Billups Rothenberg Inc., Del Mar, CA, USA) containing 5% CO 2 , 94% N 2 , and 1% O 2 , which reflects the in vivo environment. The NP cells were seeded into dishes and incubated in DMEM at 37 • C until approximately 90% confluence was reached on the following day. The medium was replaced and was supplemented with or without hydrogen peroxide (H 2 O 2 ). Thioflavin-S (NSC71948; Sigma-Aldrich, St. Louis, MO, USA) is a small molecule, chemical inhibitor of the interaction between Bag-1 and heat shock proteins, including HSC70, HSP70, and Raf-1 kinase [31,32]. NP cells were treated with 100 µM thioflavin-S at approximately 80% confluent for 16 h and the medium was replaced and supplemented with or without H 2 O 2 .

Bag-1 Overexpression
The pIRES2-AcGFP1 plasmid (Clontech Laboratories, Mountain View, CA, USA), contains the internal ribosome entry site of the encephalomyocarditis virus between the multiple cloning site, the kanamycin/neomycin resistance gene, and the Aequorea coerulescens green fluorescent protein (AcGFP) coding region. The cDNA of rat Bag-1 (GenBank NM_001106647. 3) covering the entire open reading frame was cloned into the pIRES2-AcGFP1 plasmid. NP cells were seeded into 6-well plates at approximately 80% confluence 24 h before transfection. NP cells were transfected with the Bag-1 expression plasmid using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) reagent. Data were collected by BD LSR Fortessa (Beckton Dickinson, San Jose, CA, USA) and analyzed with FlowJo version 10.8.1 software (Beckton Dickinson). From after 24-48 h of transfection, the cells were incubated with 800 µg/mL of G418 (Nacalai Tesque)-containing medium for selection from over 7-10 days. The transfected cells were seeded onto new plates and used for subsequent experiments.

Immunohistological Studies
To gain insight into the expression of Bag-1 in the IVD, freshly isolated spines from 11-week-old rats were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), decalcified and embedded in paraffin wax. Sagittal sections were deparaffinized in xylene, rehydrated through a graded ethanol series and incubated with antibodies against Bag-1 (#ab32109; Abcam plc, Cambridge, UK) in 2% normal goat serum in tris buffered saline with Tween 20 (TBS-T) at a 1:50 dilution overnight at 4 • C. The sections were washed thoroughly and then stained with N-Histofine ® Simple Stain™ Rat MAX PO (NICHIREI BIOSCIENCES INC. Tokyo, Japan). Nuclei were stained with haematoxylin. The color was developed using 3, 30-diaminobenzidine (Vectastain Universal Quick Kit; Vector Laboratories Inc., Newark, CA, USA) and the slides were examined under a microscope (BX63; Olympus, Tokyo, Japan).

Cell Viability Assay
The NP cells were seeded into 96-well plates and incubated in DMEM at 37 • C at approximately 90% confluence, treated with 200 and 400 µM of H 2 O 2 in DMEM medium, and incubated 37 • C for 24 h. Cell viability was evaluated using the Cell Counting Kit-8 assay (CCK-8; Dojindo, Kumamoto, Japan) following the manufacturer's protocol. Measurements at 450/650 nm were made 2 h after the addition of CCK-8 reagent using a SpectraMax i3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).

Measurement of Intracellular ROS
Intracellular ROS levels were assessed using fluorogenic probes with the ROS Assay Kit-Photo-oxidation Resistant DCFH-DA-(R253; Dojindo) following the manufacturer's protocol. The cells were incubated for 30 min at 37 • C with the ROS Assay working solution and then treated with H 2 O 2 . After washing twice with DMEM, the cells were fixed with 4% paraformaldehyde and Hoechst 33342 (Dojindo) was added to detect the nuclei. The stained cells were observed by fluorescence microscopy (Axio Imager M2; Carl ZEISS AG, Oberkochen, Germany) at 550/605nm (excitation/emission). The fluorescence intensity derived from DCFH-DA was calculated as the average intensity value of the stained cells by ZEISS ZEN 3.2 (blue edition) software (Carl ZEISS AG).

Detection of Mitochondrial Membrane Potential
To assess mitochondrial function, the mitochondrial membrane potential was measured using the MT-1 MitoMP Detection Kit (MT-1; Dojido) following the manufacturer's protocol. Cells were incubated with MT-1 working solution for 30 min at 37 • C, followed by treatment with H 2 O 2 . After two washes with DMEM, the cells were fixed with 4% paraformaldehyde and Hoechst 33342 was added to detect the nuclei. The stained cells were observed by fluorescence microscopy (Axio Imager M2; Carl ZEISS AG) at 470/525nm (excitation/emission). The fluorescence intensity derived from MT-1 was calculated as the average intensity value of the stained cells by ZEISS ZEN 3.2 (blue edition) software (Carl ZEISS AG).

Real-Time RT-PCR Analysis
Total RNA was extracted from NP cells using RNAeasy mini columns (Qiagen, Hilden, Germany). Before elution, the RNA was treated with RNase-free DNase I (Qiagen). The purified, DNA-free RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). Template cDNA and genespecific primers were added to Power SYBR Green Master Mix (Applied Biosystems) and mRNA expression was quantified using the Step One Plus Real-time PCR System (Applied Biosystems). β-actin was used to normalize gene expression. Melting curves were analyzed to verify the specificity of the PCR product and the absence of primer-dimers.

Statistical Analysis
All measurements were performed at least three times and the data are presented as the mean ± standard deviations (SD). Differences between groups were analyzed using the Student's t-test or Tukey-Kramer test. Statistical significance was set at p < 0.05.

Evaluation of Bag-1 Expression in NP Cells
To confirm the expression of Bag-1 in NP tissue, we stained sections of rat discs with antibodies against Bag-1 ( Figure 1A,B). The results showed prominent expression of Bag-1 in NP tissue with many cells.
The average transfection efficiency of Bag-1 plasmid into NP cells was 25.1% at 48 h after transfection ( Figure 1C). Next, to measure the expression of Bag-1 in cells after selection, real-time PCR was used to quantitate Bag-1 mRNA in NP cells and in NP cells transfected with the Bag-1 gene. Bag-1 mRNA was significantly increased in Bag-1transfected NP cells compared with untreated NP cells ( Figure 1D). Similarly, quantitation of immunoblots prepared with Bag-1-transfected cell extracts revealed a significant increase in Bag-1 protein compared with untreated NP cells ( Figure 1E). These findings indicate that Bag-1-transfected and antibiotic-selected NP cells stably express Bag-1 mRNA and these NP cells may be considered Bag-1-overexpressing cells compared with normal NP cells.

H2O2 Impaired NP Cell Viability and Mitochondrial Function and Increased Intracellular ROS
Cell viability was measured to determine the effects of H2O2 on NP cells from 0-400 μM. CCK-8 assays revealed that the groups treated with between 200 and 400 μM H2O2 for 24 h exhibited significantly decreased cell viability compared with the control ( Figure  2A). The production of intracellular reactive oxygen spices (ROS) increased significantly with H2O2 treatment in a dose-dependent manner ( Figure 2B,C). Conversely, the analysis of mitochondrial membrane potential using MT-1 dye revealed red fluorescence without H2O2 treatment, whereas H2O2 treatment resulted in a significant decrease in the intensity of MT-1 derived fluorescence ( Figure 2B,D).

H 2 O 2 Impaired NP Cell Viability and Mitochondrial Function and Increased Intracellular ROS
Cell viability was measured to determine the effects of H 2 O 2 on NP cells from 0-400 µM. CCK-8 assays revealed that the groups treated with between 200 and 400 µM H 2 O 2 for 24 h exhibited significantly decreased cell viability compared with the control (Figure 2A). The production of intracellular reactive oxygen spices (ROS) increased significantly with H 2 O 2 treatment in a dose-dependent manner ( Figure 2B,C). Conversely, the analysis of mitochondrial membrane potential using MT-1 dye revealed red fluorescence without H 2 O 2 treatment, whereas H 2 O 2 treatment resulted in a significant decrease in the intensity of MT-1 derived fluorescence ( Figure 2B

Bag-1-Overexpressing NP Cells Attenuate the Effect of H2O2 on Cell Viability, Mitochondrial Function, and Increased Intracellular ROS Levels
The viability of Bag-1-overexpressing NP cells by CCK-8 analysis did not show a significant change under 200 μM H2O2 treatment for 24 h compared with the untreated group, whereas a significant decrease was observed under 400 μM H2O2 treatment compared with the untreated group and 200 μM H2O2 treatment for 24 h ( Figure 3A). The viability of Bag-1-overexpressing NP cells was significantly higher compared with that of NP cells treated with similar H2O2 concentrations ( Figure 3B). The level of intracellular ROS production increased significantly with H2O2 treatment at 200 and 400 μM for 24 h compared with the untreated group ( Figure 3C,D). Moreover, the mitochondrial membrane potential was not significantly altered in cells treated with H2O2 ( Figure 3C,E).

Bag-1 Binds to HSP70 in NP Cells, but Does Exhibit Obvious Raf-1 Binding
Previous studies have demonstrated an interaction between Bag-1, HSP70, and Raf-1 in various cells [13,15,19,20,[25][26][27]33]. Therefore, we evaluated this putative proteinprotein binding in NP cells. NP cell extracts were immunoprecipitated with anti-Bag-1 antibody and the direct interaction partners were detected by immunoblotting using specific antibodies. As expected, Bag-1 was bound to HSP70 and these complexes were

Bag-1 Binds to HSP70 in NP Cells, but Does Exhibit Obvious Raf-1 Binding
Previous studies have demonstrated an interaction between Bag-1, HSP70, and Raf-1 in various cells [13,15,19,20,[25][26][27]33]. Therefore, we evaluated this putative proteinprotein binding in NP cells. NP cell extracts were immunoprecipitated with anti-Bag-1 antibody and the direct interaction partners were detected by immunoblotting using spe-cific antibodies. As expected, Bag-1 was bound to HSP70 and these complexes were evident following treatment with between 200 and 400 µM H 2 O 2 for 24 h ( Figure 4A). Similarly, immunoprecipitation of HSP70 resulted in co-precipitation of Bag-1 under all conditions ( Figure 4A). In contrast, immunoprecipitation of Bag-1 in NP cells did not result in coprecipitation of Raf-1 under all conditions ( Figure 4B). Additionally, we only detected an exceedingly faint expression of Bag-1 by immunoprecipitation of Raf-1 following treatment with between 200 and 400 µM H 2 O 2 ( Figure 4B).  Figure 4A). Similarly, immunoprecipitation of HSP70 resulted in co-precipitation of Bag-1 under all conditions ( Figure 4A). In contrast, immunoprecipitation of Bag-1 in NP cells did not result in co-precipitation of Raf-1 under all conditions ( Figure 4B). Additionally, we only detected an exceedingly faint expression of Bag-1 by immunoprecipitation of Raf-1 following treatment with between 200 and 400 μM H2O2 ( Figure 4B).

Treatment of Bag-1 with an Inhibitor of the Binding Site for HSP70 and Raf-1 Attenuates NP Cell Viability, Mitochondrial Function, and Increased Intracellular ROS Levels
Thioflavin-S (NSC71948) is an inhibitor of the interaction between Bag-1 and HSP70 or Raf-1 [31,32]. This compound selectively inhibits Bag-1 binding through the C-terminal binding site of HSP70 or Raf-1 [32]. To determine whether Bag-1-HSP70 binding is involved in protecting NP cells from oxidative stress, NP cells were incubated with thioflavin-S and cell viability, ROS accumulation, and mitochondrial activity were measured following H2O2 treatment. The cell viability of NP cells decreased markedly following treatment with between 200 and 400 μM H2O2 for 24 h ( Figure 5A). The viability of NP cells was significantly higher compared with that of thioflavin-S-treated NP cells at similar H2O2 concentrations ( Figure 5B). ROS accumulation in thioflavin-S-treated NP cells was increased significantly following between 200 and 400 μM H2O2 treatment for 24 h in a dose-dependent manner ( Figure 5C,D). The mitochondrial membrane potential was significantly decreased based on the intensity of MT-1-derived fluorescence following H2O2 treatment ( Figure 5C,E).

Treatment of Bag-1 with an Inhibitor of the Binding Site for HSP70 and Raf-1 Attenuates NP Cell Viability, Mitochondrial Function, and Increased Intracellular ROS Levels
Thioflavin-S (NSC71948) is an inhibitor of the interaction between Bag-1 and HSP70 or Raf-1 [31,32]. This compound selectively inhibits Bag-1 binding through the C-terminal binding site of HSP70 or Raf-1 [32]. To determine whether Bag-1-HSP70 binding is involved in protecting NP cells from oxidative stress, NP cells were incubated with thioflavin-S and cell viability, ROS accumulation, and mitochondrial activity were measured following H 2 O 2 treatment. The cell viability of NP cells decreased markedly following treatment with between 200 and 400 µM H 2 O 2 for 24 h ( Figure 5A). The viability of NP cells was significantly higher compared with that of thioflavin-S-treated NP cells at similar H 2 O 2 concentrations ( Figure 5B). ROS accumulation in thioflavin-S-treated NP cells was increased significantly following between 200 and 400 µM H 2 O 2 treatment for 24 h in a dose-dependent manner ( Figure 5C,D). The mitochondrial membrane potential was significantly decreased based on the intensity of MT-1-derived fluorescence following H 2 O 2 treatment ( Figure 5C,E). The upper row of the figure represents ROS production, which was monitored using R253 (green) and the lower row is the mitochondrial membrane potential, which was monitored using MT-1 (orange). The nucleus was detected by Hoechst 33342 reagent (blue). The detection of ROS production increased under H2O2 treatment, and the detection of MT-1 decreased. Scale bars = 20 μm. (D) Quantitative data of ROS production (R253) in thioflavin-S-treated NP cells. Data are the means ± SD n = 3, * p < 0.05, ** p < 0.05. (E) Quantitative data of the mitochondrial membrane potential (MT-1) in thioflavin-S-treated NP cells. Data are the means ± SD n = 3, * p < 0.05.

The Effects of Bag-1 on MAPKs and Raf-1 Activation
To further determine the effects of the putative Bag-1, HSP70, and Raf-1 interactions on MAPK kinase activity (p38, ERK1/2 and JNK), we measured the phosphorylation status The upper row of the figure represents ROS production, which was monitored using R253 (green) and the lower row is the mitochondrial membrane potential, which was monitored using MT-1 (orange). The nucleus was detected by Hoechst 33342 reagent (blue). The detection of ROS production increased under H 2 O 2 treatment, and the detection of MT-1 decreased. Scale bars = 20 µm. (D) Quantitative data of ROS production (R253) in thioflavin-S-treated NP cells. Data are the means ± SD n = 3, * p < 0.05, ** p < 0.05. (E) Quantitative data of the mitochondrial membrane potential (MT-1) in thioflavin-S-treated NP cells. Data are the means ± SD n = 3, * p < 0.05.

The Effects of Bag-1 on MAPKs and Raf-1 Activation
To further determine the effects of the putative Bag-1, HSP70, and Raf-1 interactions on MAPK kinase activity (p38, ERK1/2 and JNK), we measured the phosphorylation status of p38, ERK1/2, JNK, and Raf-1 following H 2 O 2 treatment by Western blot analysis. NP cells were treated with between 200 and 400 µM H 2 O 2 under hypoxic conditions for 30 min. The phosphorylation of Raf-1 did not increase significantly with 200 µM H 2 O 2 treatment but increased significantly with 400 µM H 2 O 2 treatment compared with 0 µM H 2 O 2 ( Figure 6A-C). The phosphorylation of p38 and ERK1/2 in NP cells increased significantly with between 200 and 400 µM H 2 O 2 treatment compared with untreated cells (Figure 6A-C).
Similarly, in Bag-1-overexpressing cells, the phosphorylation of Raf-1 did not significantly increase at 200 μM H2O2 but increased significantly at 400 μM H2O2 compared with the untreated cells ( Figure 7A-C). Unlike NP cells, the phosphorylation of p38 in Bag-1overexpressing cells did not significantly change following treatment with between 200 and 400 μM H2O2 ( Figure 7B). In Bag-1 overexpressing NP cells, the phosphorylation of ERK1/2 in-creased significantly following treatment with between 200 and 400 μM H2O2 compared with the untreated cells ( Figure 7B,C). In untreated Bag-1-overexpressing NP cells, the phosphorylation of ERK1/2 did not significantly change compared with NP cells ( Figure 7D).
In thioflavin-S-treated NP cells, the phosphorylation of p38 increased significantly following treatment with between 200 and 400 μM H2O2 treatment compared with untreated cells and the phosphorylation of Raf-1 increased significantly at 400 μM H2O2, although the phosphorylation of ERK1/2 did not change significantly under all conditions ( Figure 8A-C). Without H2O2, the phosphorylation of ERK1/2 in thioflavin-S treated NP cells was significantly increased compared with NP cells ( Figure 8D).
In addition, the phosphorylation of JNK in NP cells, Bag-1 overexpressing NP cells, and thioflavin-S-treated NP cells showed no significant changes under all conditions (Figures 6-8B,C).  Similarly, in Bag-1-overexpressing cells, the phosphorylation of Raf-1 did not significantly increase at 200 µM H 2 O 2 but increased significantly at 400 µM H 2 O 2 compared with the untreated cells ( Figure 7A-C). Unlike NP cells, the phosphorylation of p38 in Bag-1-overexpressing cells did not significantly change following treatment with between 200 and 400 µM H 2 O 2 ( Figure 7B). In Bag-1 overexpressing NP cells, the phosphorylation of ERK1/2 in-creased significantly following treatment with between 200 and 400 µM H 2 O 2 compared with the untreated cells ( Figure 7B,C). In untreated Bag-1-overexpressing NP cells, the phosphorylation of ERK1/2 did not significantly change compared with NP cells ( Figure 7D).
In thioflavin-S-treated NP cells, the phosphorylation of p38 increased significantly following treatment with between 200 and 400 µM H 2 O 2 treatment compared with untreated cells and the phosphorylation of Raf-1 increased significantly at 400 µM H 2 O 2 , although the phosphorylation of ERK1/2 did not change significantly under all conditions ( Figure 8A-C). Without H 2 O 2 , the phosphorylation of ERK1/2 in thioflavin-S treated NP cells was significantly increased compared with NP cells ( Figure 8D).     In addition, the phosphorylation of JNK in NP cells, Bag-1 overexpressing NP cells, and thioflavin-S-treated NP cells showed no significant changes under all conditions (Figures 6, 7 and 8B,C).

Discussion
Previous studies on the aging of vertebrate discs reported that H 2 O 2 induces excessive ROS production, which leads to oxidative stress and damaged cells. Mitochondria also suffer damage and dysfunction as evidenced by the loss of mitochondrial membrane potential [5,8]. Mitochondrial dysfunction is associated with aging and degenerative disease progression, which results in cell damage as a secondary consequence of oxidative stress [34,35]. Several recent studies have reported that interventions targeting oxidative stress protect NP cells from damage and cell death, thus, preventing IVD degeneration [5][6][7][8][9].
The MAPK signaling pathway is an important pathway involved in ROS-triggered cell damage in NP cells [5,7,8,12,34,36]. In the present study, H 2 O 2 treatment increased NP cell death and upregulated the phosphorylated p38 and ERK levels. It was reported that p38 activation is a marker of senescence and induces the senescence-associated secretory phenotype [2,37]. Other studies on NP cells have demonstrated H 2 O 2 -induced oxidative stress through MAPK signaling pathways, particularly p38 [5,12,[38][39][40]. Thus, MAPK activation effectively suppresses the accumulation of ROS and induces cell senescence.
Bag-1 protects cells and tissues against various stresses, such as oxidative stress, by interacting with HSP70 in response to stressors [14,18,25,33]. HSP70 contains a nucleotidebinding domain (NBD) where ATP is bound and hydrolyzed [15]. The C-terminus of Bag-1 binds to the NBD of HSP70 and acts as a nucleotide-exchange factor; this interaction assists HSP70 function as a chaperone [15,33,41]. Bag-1 overexpression increases cell viability and protects cells by upregulating HSP70 activity [14,17,25], which suggests that Bag-1 stabilizes HSP70 protein in cells [14]. HSP70 regulates the quality of intracellular protein folding and contributes to cell survival under various conditions [41]. Furthermore, overexpression of HSP70 suppresses ROS production from the mitochondria [22] and regulates mitochondrial function to adapt NP cells to various stresses [10].
In the present study, H 2 O 2 induced intracellular ROS levels in Bag-1-overexpressing NP cells, but mitochondrial function did not decrease, whereas NP cells treated with thioflavin-S exhibited increased ROS accumulation and decreased mitochondrial function and cell viability. In addition, our findings indicated that Bag-1 binds to HSP70, whereas we did not clearly observe binding between Bag-1 and Raf-1 under normal conditions or after H 2 O 2 treatment. Raf-1 immunoprecipitation showed only a slight co-precipitation band under H 2 O 2 treatment; however, it is possible that Raf-1-Bag-1 binding may gradually increase during stress intensity. Although it is difficult to precisely quantitate the amount of Bag-1-HSP70 and Bag-1-Raf-1 binding, our results indicate that Bag-1 preferentially binds to HSP70 compared with Raf-1 and the inhibitor of the Bag-1 C-terminus primarily influences the interaction of Bag-1 and HSP70 in NP cells. The finding that Bag-1-HSP70 binding was clearly observed in NP cells, but Bag-1-Raf-1 was not under normal conditions suggest the possibility that Bag-1-HSP70 binding enhances HSP70 function to maintain intracellular homeostasis and mitochondrial function in NP cells. These effects are considered to protect NP cells against oxidative stress induced by H 2 O 2 . Moreover, these Bag-1-mediated protective effects suppress p38 phosphorylation in NP cells and downregulate the p38/MAPKs signaling pathway, which enhances NP cell viability. The regulation of p38 activity induces the senescence-associated secretory phenotype [2,37], which reduces NP cell senescence.
With respect to Bag-1 and Raf-1, it has been reported that Bag-1 and Ras each activate Raf-1 independently [15,27]. Subsequently, Bag-1-Raf-1 binding activates signaling molecules including ERK, which attenuates apoptosis [17,25,42]. In contrast, IVD studies indicate that the ERK/MAPK signaling pathway is activated by excessive ROS in IVD cells and promotes IVD degeneration, including extracellular matrix reduction, apoptosis, and cell damage [36,38,39]. Thus, the inhibition of ERK signaling is considered a potential treatment for IVD degeneration as it provides some protection against the adverse effects of TNF-α in the IVD [43]. Meanwhile, hypoxia-induced ERK phosphorylation and ERK activation was shown to be necessary for NP cell survival under hypoxic conditions [44]. Therefore, excessive suppression of ERK is also a disadvantage to NP cell survival. We evaluated NP cells under hypoxic conditions (1% O 2 ), which reflects the in vivo environment, although culture conditions may affect the ERK response. When we inhibited the C-terminus of Bag-1 in NP cells, ERK phosphorylation was increased compared with normal NP cells, even under H 2 O 2 -free conditions, whereas Bag-1 overexpression did not increase ERK phosphorylation compared with normal NP cells under H 2 O 2 -free conditions. Although the increase in Raf-1 phosphorylation by H 2 O 2 treatment showed a similar response in all NP cell groups, treatment with the Bag-1 C-terminal inhibitor did not alter ERK phosphorylation with or without H 2 O 2 stimulation. There are few studies regarding an independent analysis of Raf-1 in NP cells; however, our findings suggest that the Bag-1-Raf-1 interaction is not so important to ERK activation, and that Raf-1 is regulated by different factors, such as Ras, rather than Bag-1 in NP cells. Based on previous reports of ERK in NP cells, if Bag-1 enhances ERK activation via Raf-1, it is not beneficial for NP cell protection. Therefore, our results, which did not show a Bag-1-Raf-1 interaction, do not contradict the cell protective effects of Bag-1.
The present study suggests the following two possibilities: (1) Bag-1 preferentially interacts with HSP70, rather than Raf-1, to protect NP cells against oxidative stress; (2) the effect of Bag-1 on Raf-1 may be less compared with that of HSP70 in NP cells. These possibilities are supported by the finding that NP cells, which exhibited inhibited Bag-1-HSP70 binding, had decreased cell viability and mitochondrial function under H 2 O 2 treatment.
We also did not observe significant changes in JNK/MAPKs signaling under the conditions of this study. There are the reports that oxidative stress, such as H 2 O 2 or tert-butyl hydroperoxide (t-BHP), provoke JNK phosphorylation in disc cells, and HSP70 has been reported to downregulate the JNK/c-Jun pathway [11,12]. These discrepancies presumably result from differences in the types of oxidative stress-inducing agents, administration conditions, and cell culture conditions.
Our study had some limitations. This study focused on the protective ability of Bag-1 and whether Bag-1, HSP70, and Raf-1 interacted in NP cells. Thus, other signaling pathways or factors that have been reported to be involved, such as MAPKs, HSP70, Raf kinases, other Bag-1 interaction partners, such as Bcl-2, and mitochondrial function, were not evaluated in this study. To quantitate and compare the binding of HSP70 or Raf-1 was also difficult. We have concluded that Bag-1-HSP70 contributes to maintaining mitochondrial function; however, there is a possibility that mitochondrial HSP70 (e.g., mtHSP70) interacts with Bag-1, which remains unclear and requires further investigation.
In conclusion, we demonstrated, for the first time, that Bag-1 protects NP cells from oxidative stress by interacting with HSP70. We also showed that Bag-1 primarily interacts with HSP70, rather than Raf-1, in NP cells. Bag-1 suppressed mitochondrial dysfunction and p38/MAPKs activation under oxidative stress conditions, resulting in NP cell survival. However, additional studies are needed to confirm the effects of Bag-1 and its potential interaction with other intracellular signaling factors that may play an important role in understanding the pathogenesis of intervertebral disc degeneration and its treatment.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.