Mechanism of the Mitogen-Activated Protein Kinases/Mammalian Target of Rapamycin Pathway in the Process of Cartilage Endplate Stem Cell Degeneration Induced by Tension Load

Isolation, Extraction, and Culture of Rabbit CESCs

Two specific pathogen-free female white big-eared rabbits (6 months old) from New Zealand were euthanized by intravenous injection of pentobarbital sodium (200 mg/kg) at the ear margin. ²⁶ The fur on the back was removed followed by disinfection with 75% alcohol. The rabbit thoracolumbar spine was removed through the back approach and immediately transferred to a sterile ultra-clean workbench. The T10-L5 IVD segment was collected, and then the surrounding attached muscles, ligaments, and bone fragments were carefully removed and rinsed with phosphate buffer saline (PBS) buffer containing 1000 U/mL penicillin and 1 g/L streptomycin (Gibco, NY, USA). Under the anatomical microscope (SZX16, Olympus, Japan), the annulus fibrosus was incised with a blade, and the nucleus pulposus and annulus fibrosus were scraped away. The CEP with thin middle, thick edge, and transparent shape was put into the petri dish. After washing with the above PBS solution, the CEP was cut into 1 mm³ pieces, detached with 0.25% ethylenediaminetetraacetic acid (EDTA)-trypsin solution (Gibco) and 0.2% type II collagenase (Gibco), and then filtered through a sieve, and endplate chondrocytes were obtained by centrifugation at 600 g for 8 minutes. Cell suspension was prepared with Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Gibco) containing 20% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and cultured at 37˚C with 5% CO₂ to obtain primary endplate chondrocytes.

Stem cells were screened by the principle that low-melting point agarose stem cells can form cell clusters in the suspension culture system.^27,28 Agarose (Bio-Rad, Hercules, CA, USA) was dissolved in a PBS solution to obtain agarose at concentrations of 20 g/L and 40 g/L. Agarose (20 g/L) was placed on the bottom layer of the petri dish and solidified at 4°C for 1 hour. Cell suspension (5 × 10⁴ cells/mL) and an equal volume of agarose (40 g/L) were mixed into the petri dish (10% FBS), and condensed at 4˚C for 15 minutes, followed by culture at 37˚C with 5% CO₂, and the medium was changed every 2-3 days. Cell clusters formed after 6 weeks and were selected by cell cloning screening system ClonePix2 (Genetix, Molecular Devices, UK) and transferred to 12-well plates and cultured in DMEM/F12 medium (20% FBS) at 37°C with 5% CO₂ for amplification in vitro. The obtained cells were subjected to subsequent experimentation.

Flow Cytometry

The well-growing CESCs with a cell density of 80% were prepared into 2 × 10⁶/mL cell suspensions. Cell suspension (500 μL) was incubated with 5 μL monoclonal antibodies FITC-fluorescein-conjugated anti-CD90 (anti-CD90-FITC, ab124527, Abcam), anti-CD105 (anti-CD105-FITC, ab18278, Abcam), and anti-CD34 (anti-CD34-FITC, ab131589, Abcam) in the dark for 20 minutes, with mouse homotype antibodies as control. After the cells were washed in PBS and re-suspended, the cells were detected by flow cytometry immediately.

Identification of Multidirectional Differentiation Ability of CESCs

The CESCs obtained by monoclonal methods were induced to differentiate into adipogenic, osteogenic, and chondrogenic types, respectively. The steps of adipogenic induction and differentiation were as follows ²⁹ : the cells were detached with 0.25% EDTA-trypsin and seeded into 24-well plates at 2 × 10⁴ cells/well, and the adipogenic differentiation medium (DMEM/F12: 10% FBS, 500 μmol/L methylisobutylxanthine, 1 μmol/L dexamethasone, 60 μmol/L indomethacin, and 5 mg/L insulin) was added to induce adipogenic differentiation for 3 days when the cell confluence reached 80-90%. Then the medium was replaced with the maintenance adipogenic differentiation medium (DMEM/F12: 10% FBS, 10 mg/L insulin) for induction of 1 day, and then replaced with adipogenic differentiation medium for induction of 3 days. After 3 cycles, the CESCs were fixed with 4% paraformaldehyde (Hushi, Sinopharm, Shanghai, China), stained with oil red O (Solarbio, Shanghai, China), and observed under a microscope (TS100, Nikon, Japan) and photographed.

The steps of osteogenic induction and differentiation were as follows ³⁰ : after detachment with 0.25% EDTA-trypsin, the cells were seeded into 24-well plates at 2 × 10⁴ cells per well. After 24 h of cell adherence, the medium was replaced with the osteogenic induction differentiation medium (DMEM/F12: 10% FBS, 100 nmol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 50 μmol/L L-ascorbate-2-phosphate), and changed every 3 days. After 2-3 weeks of continuous induction, the CESCs were fixed with 4% paraformaldehyde, stained with 1% alizarin red (Solarbio), and observed under a microscope and photographed.

The steps of chondrogenic induction and differentiation were as follows ³¹ : after detachment with 0.25% EDTA-trypsin, the cells were seeded into 24-well plates at 2 × 10⁴ cells/well. The cartilage induction differentiation medium (DMEM/F12: 10% FBS, 1% ITS, 100 nmol/L dexamethasone, 50 mg/L L-ascorbate-2-phosphate, and 10 μg/L recombinant human TGFβ1) was added when the cell confluence reached 80% and changed every 3 days. After 3 weeks of induction, cells were fixed with 4% paraformaldehyde and stained with 1% toluidine blue (Solarbio).

Establishment of CESCs Degeneration Models Induced by TL

The identified rabbit CESCs were subcultured. The cells at the second generation were seeded into BioFlex^TM 6-well plate (Flexcell International Corporation, Hillsborough, NC, USA) coated with type I collagen on the surface at 1 × 10⁵ cells/well for culture at 37°C with 5% CO₂. When reaching confluence 80-90%, the cells were pretreated with serum-free DMEM for 24 hours. FX-4000T cell stress loading system (Flexcell International Corporation) was used to apply periodic tension strain at 10% elongation and .5 Hz to CESCs for 4 hours/day. The internal environment of the stress loading system was a cell incubator containing 5% CO₂ at 37°C. The cells in the control group were seeded in BioFlex^TM 6-well plate with the same cell density and continued to be placed in the cell incubator for normal culture without receiving stretching. After 25 days of culture, cells were collected by trypsin digestion for subsequent experimentation.

Cell Treatment and Grouping

The CESCs were cultured in DMEM/F12 medium containing 20% FBS and 5% CO₂ at 37°C. The CESCs were divided into (1) control group: CESCs without receiving stretching (NC) (as a control for cells subjected to stretch tension); (2) Tension load group (TL): 0.5 hz and 10% of stretch tension was applied to cells for continuous treatment of 25 days at 4 h/d during cultivation; (3) TL + PBS group: the equal amount of PBS was added to the medium (as a control for cells added with rapamycin), 0.5 hz and 10% of stretch tension were applied for 25 days at 4 h/d; (4) TL + rapamycin group (TL + Rapa): 100 nM rapamycin was added to the medium (553211, Sigma-Aldrich, USA), ³² 0.5 hz and 10% of stretch tension were applied for 25 days at 4 h/d; (5) TL + silencing (si)RNA-NC group (TL + si-NC): after transfection of control siRNA (as a control for cells transfected with si-JNK and si-ERK), the cells were subjected to a stretch force of 0.5 hz and 10% for 25 days at 4 h/d; (6) TL + JNK-siRNA group (TL + si-JNK): after transfection with JNK-siRNA, the cells were subjected to a stretch force of 10% and 0.5 Hz for 25 days at 4 h/d; and (7) TL + ERK-siRNA group (TL + si-ERK): after the cells were transfected with ERK-siRNA, a stretch force of 0.5 Hz and 10% was applied for 25 days at 4 h/d days.

Cell Transfection

Well-grown CESCs with a cell density of 80% were transfected with JNK-siRNA (#6232, Cell Signaling Technology, Boston, USA), ERK-siRNA (#6560, Cell Signaling Technology), and negative control SignalSilence® Control siRNA (#6568, Cell Signaling Technology) according to the instructions of Lipofectamine™2000 transfection kit (Gibco). The culture medium was replaced 72 hours later, and the cells were collected for subsequent experimentation. Cell transfection efficiency was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide Assay

According to the instructions of MTT assay kits (Sigma-Aldrich, MO, USA), cell suspension was prepared from cells in the logarithmic growth stage and laid in the middle well of 96-well plates. The edge wells were filled with sterile PBS and the cells were cultured at 37°C with 5% CO₂ for 24 hours. MTT solution (5 mg/mL) was added to each well and cultured at 37°C for 4 hours. Then cells were added with 150 μL dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and mixed evenly to make the crystals fully dissolved. Optical density (OD) value was measured at 490 nm using a spectrophotometer (Bio-Rad 680, Bio-Rad, Hercules, CA, USA). Cell survival rate = [experimental group OD - blank group OD]/(control group OD - blank group OD) × 100%.

Annexin V-FITC/PI Double Staining

CESCs in the logarithmic growth stage were detached with 0.25% trypsin and re-suspended with PBS solution according to the instructions of Annexin V-FITC/propidium iodide (PI) apoptosis kit (YEASEN Biotech, Shanghai, China). Annexin V-FITC (5 μL) and PI (10 μL) staining solutions were added into 100 μL 1 × Binding Buffer, mixed gently, and reacted at room temperature to avoid light for 15 minutes. Flow cytometry (MoFloAstrios EQ, Beckman Coulter, Inc, CA, USA) was used for observation and detection within 1 hour.

Western Blot

Radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Sigma-Aldrich) was added into the cells and mixed. Cells were lysed on ice for 30 minutes, centrifuged at 1600 × g for 10 minutes, and the protein concentration was measured using bicinchoninic acid protein quantitative kits (Boster, Wuhan, China). Then the protein was loaded for boiling, isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electrically transferred to polyvinylidene difluoride membranes, which were sealed with 5% bovine serum albumin for 2 hours at room temperature to block the non-specific binding. Next, the membranes were incubated with anti-mTOR (ab134903, 1:1000, Abcam), anti-p-mTOR (Ser2448) (#5536, Cell Signaling Technology), anti-Raptor (ab40768, 1:1000, Abcam), anti-p-Raptor(Ser792) (#8914, Cell Signaling Technology), anti-ERK (ab32537, 1:1000, Abcam), anti-p-ERK (Thr202/Tyr204) (#9101, Cell Signaling Technology), anti-JNK (ab110724, 1:1000, Abcam), anti-p-JNK (Thr183/Tyr185) (#4668, Cell Signaling Technology), anti-p38 (ab170099, 1:1000, Abcam), anti-p-p38 (Thr180/Tyr182) (#4511, Cell Signaling Technology), anti-LC3 II/I (ab128025, Abcam), anti-Beclin-1 (ab207612, Abcam), and anti-p62 (ab109012, Abcam) at 4°C overnight. After washing the membrane, goat anti-rabbit IgG labeled with horseradish peroxidase (ab205718, 1:2000, Abcam) was added for incubation for 2 hours. Finally, the developing images were collected by ImageJ software (NIH, Bethesda, MD, USA), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference.

RT-qPCR

Total RNA was extracted from CESCs using TRIzol kits (R&D Systems Inc., MN, USA), and cDNA was synthesized with 1 μg RNA as a template using reverse transcription kits (Takara Bio Inc., Dalian, China). Gene fragments were amplified using cDNA as a template. PCR reaction system (20 μL) included cDNA template 2 μL, SYBR Premix Ex Taq II (Takara) 10 μL, forward and reverse primers 1 μL each, and supplemented to 20 μL with ddH₂O. Reaction conditions: 94°C for 10 s followed by 40 cycles (95°C for 5 s; 55°C for 35 s; and 72°C for 40 s); final extension at 4°C for 10 minutes. With GAPDH as an internal reference, the relative expression level was calculated based on the 2^-△△ct method. All RT-qPCR data were normalized to GAPDH for quantitative comparison. All RT-qPCR primers (Table 1) were synthesized by Sangon Biotech (Shanghai, China).

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Table 1.

Primer Sequence of RT-qPCR.

Gene	Primer sequences
ERK F	5′-TACGTACGTCCTCCCCTCAC-3′
ERK R	5′- GAGAGCCTGTCGTTCCTGAC-3′
JNK F	5′-CAGACAGCAGGTATGCCAGA-3′
JNK R	5′-TACCCAAAACGCAAAGTTCC-3′
Aggrecan F	5′-GCTACGGAGACAAGGATGAGTTC-3′
Aggrecan R	5′-CGTAAAAGACCTCACCCTCCAT-3′
COL-2A F	5′-AACACTGCCAACGTCCAGAT-3′
COL-2A R	5′-CTGCAGCACGGTATAGGTGA-3′
SOX9 F	5′-TCTACCGCTGTGAGGTGATGC-3′
SOX9 R	5′-TTCACCACGACCTCCAAGG-3′
GAPDH F	5′-CTCAACTACATGGTCTACATGTTCCA-3′
GAPDH R	5′-CTTCCCATTCTCAGCCTTGACT-3′

Statistical Analysis

SPSS 21 (IBM Corp. Armonk, NY, USA) and GraphPad Prism 8.01 (GraphPad Software Inc., San Diego, CA, USA) were used for statistical analysis and plotting. The t test was used for data comparison between 2 groups. One-way analysis of variance (ANOVA) was used for data comparison among multiple groups, and Tukey’s test was used for the post hoc test. A probability value of P < .05 indicated the difference was statistically significant.

CEP Degeneration Induced by TL

First, the rabbit CESCs were isolated and extracted. Flow cytometry showed positive expression of stem cell markers CD90 and CD105 and negative expression of CD34 (Figure 1A), indicating that CESCs were successfully obtained from rabbit CEP. To further verify whether rabbit CESCs have the biological differentiation ability of stem cells, we conducted the three-way differentiation induction of adipogenesis, osteogenesis, and chondrogenesis. The results manifested that rabbit CESCs have the differentiation ability of adipogenesis, osteogenesis, and chondrogenesis (Figure 1B). These results indicate that we have successfully obtained the rabbit CESCs. To deliberate the effect of TL on CESCs, we applied the FX-4000T cell stress loading system to impose 10% elongation and 0.5 Hz periodic tension strain to cells. Cell viability was determined by MTT assay, which elicited decreased cell viability of the TL group compared with the NC group (P < .01) (Figure 1C). Annexin V-FITC/PI double staining showed higher apoptosis levels in the TL group than the NC group (P < .01) (Figure 1D). The expressions of cartilage markers proteoglycan ACAN, type II collagen COL-2A, and transcriptional regulator Sox9 were detected by RT-qPCR, which elucidated decreased ACAN, COL-2A, and SOX9 expressions in the TL group compared to the control group (all P < .01) (Figure 1E), indicating the degeneration of CESCs induced by TL.OPEN IN VIEWER

Inhibition of the mTOR Pathway Alleviated TL-Induced CEP Degeneration by Activating Autophagy

To ascertain whether the mTOR pathway is involved in the degeneration of CESC under TL, we detected the protein expressions of mTOR, p-mTOR, Raptor, and p-Raptor in the mTOR pathway by Western Blot (WB). The results elicited that the ratio of p-mTOR/mTOR and the ratio of p-Raptor/Raptor were significantly increased (all P < .01) (Figure 2A), indicating the activation of phosphorylation in the mTOR pathway.OPEN IN VIEWER

Rapamycin is frequently used as a specific inhibitor of the mTOR pathway. ³² Therefore, we further verified the influence of the mTOR pathway on the degeneration level of CESCs after TL by adding 100 nM rapamycin to the medium. Western blot showed that the ratios of p-mTOR/mTOR and p-Raptor/Raptor were lowered after the addition of rapamycin (all P < .05), indicating the suppression of the mTOR pathway (Figure 2A). Furthermore, MTT assay was used to detect cell viability, which discovered that compared with the TL + PBS group, cell viability was increased in the TL + Rapa group (P < .01) (Figure 2B). Annexin V-FITC/PI double staining showed that the apoptosis level of the TL + Rapa group was lower than the TL + PBS group (P < .01) (Figure 2C). Subsequent RT-qPCR showed that the levels of cartilage markers ACAN, COL-2A, and SOX9 in CESCs were up-regulated after adding rapamycin (all P < .05) (Figure 2D). These results suggest that inhibition of the mTOR pathway can alleviate TL-induced CEP degeneration. To figure out whether the mTOR pathway regulates CESCs degeneration through autophagy, we detected the protein levels of autophagy markers LC3-II, Beclin-1, and p62 by WB. The results demonstrated that the levels of LC3-II and Beclin-1 in the TL-Rapa group were up-regulated, and p62 was reduced (all P < .05) (Figure 2E), suggesting that the addition of rapamycin could activate autophagy of CESCs. Taken together, down-regulation of the mTOR pathway may alleviate TL-induced CEP degeneration by activating autophagy.

The MAPK Pathway Was Stimulated in CESCs After TL

Presently, cumulative studies indicate that the MAPK pathway may regulate the mTOR pathway.^24,25 First, the key proteins ERK, JNK, p38, p-ERK, p-JNK, and p-p38 of the MAPK pathway in CESCs under TL were detected by WB. Interestingly, compared with the NC group, the protein expressions of p-ERK, p-JNK, and p-p38 in the TL group were visibly enhanced (all P < .05), while there were no significant differences in protein expressions of ERK, JNK, and p38 (all P > .05) (Figure 3), indicating that the MAPK pathway was activated in CESCs after TL.OPEN IN VIEWER

JNK Knockdown Alleviated CESC Degeneration by Inhibiting Phosphorylation of Raptor

JNK, a key protein of the MAPK pathway, phosphorylates Raptor, a key regulatory part of mTORC1. ³³ The molecular association between JNK and Raptor may be a potential regulatory mechanism between the MAPK and mTOR pathways.^34,35 Therefore, we further studied the regulation of JNK and Raptor in TL-induced CESC degeneration. To begin with, si-JNK and its negative control si-JNK-NC (si-NC) were transfected into TL-treated CESCs, and the mRNA expression of JNK was detected by RT-qPCR. The results discovered that the JNK expression in TL-treated CESCs was successfully repressed after transfection with si-JNK (P < .01) (Figure 4A). Western blot showed that the p-Raptor/Raptor ratio in the TL + si-JNK group was decreased (P < .05) (Figure 4B), indicating that the JNK pathway regulates Raptor phosphorylation. To further investigate the role of JNK in regulating Raptor phosphorylation in CESCs degeneration under TL, we performed MTT assay and Annexin V-FITC/PI double staining. The results uncovered that compared with the TL + si-NC group, si-JNK transfected cells showed enhanced cell activity and decreased apoptosis level (P < .01) (Figures 4C and 4D). Subsequent RT-qPCR showed that the levels of cartilage markers ACAN, COL-2A, and SOX9 were up-regulated in the TL + si-JNK group compared to the TL + si-NC (all P < .05) (Figure 4E). These results suggest that inhibition of JNK can reduce TL-induced CEP degeneration by down-regulating Raptor phosphorylation. To investigate whether JNK-mediated phosphorylation of Raptor affects the degeneration of CESCs by regulating autophagy, we detected the protein levels of autophagy markers LC3-II, Beclin-1, and p62 by WB. The results revealed that the levels of LC3-II and Beclin-1 in the TL + si-JNK group were up-regulated and p62 was reduced (all P < .05), indicating that inhibition of JNK expression can activate autophagy of TL-treated CESCs (Figure 4F). In conclusion, down-regulation of JNK may activate autophagy by inhibiting Raptor phosphorylation, thereby alleviating TL-induced CEP degeneration.OPEN IN VIEWER

ERK Knockdown Alleviates CESC Degeneration by Inhibiting Phosphorylation of mTOR

In addition to the mechanism of JNK regulating Raptor phosphorylation, there may be many other regulatory mechanisms between the MAPK and mTOR pathways. It has been reported that ERK, a key protein of the MAPK pathway, regulates cell proliferation by mediating the phosphorylation of mTOR. ³⁶ To explore whether ERK regulates mTOR phosphorylation during CESC degeneration, we transfected TL-induced cells with si-ERK or si-ERK-NC (si-NC). The mRNA level of ERK was detected by RT-qPCR, and the results showed that the level of ERK in the TL + si-ERK group was lower than that of the TL + si-NC group (P < .05) (Figure 5A), indicating that ERK in TL-treated CESCs was successfully inhibited. Western blot showed that the ratio of p-mTOR/mTOR was obviously decreased (P < .05) (Figure 5B), indicating that ERK regulates mTOR phosphorylation in CEP degeneration models. To further explore the role of ERK in regulating the mTOR phosphorylation in the degeneration of TL-induced CESCs, we conducted MTT assay and Annexin V-FITC/PI double staining. Compared with the TL + si-NC group, the cell activity was elevated and apoptosis level was decreased in the TL + si-ERK group (Figures 5C and 5D). RT-qPCR showed up-regulated levels of cartilage markers ACAN, COL-2A, and SOX9 in the TL + si-ERK group compared with the TL + si-NC group (all P < .05) (Figure 5E). These results elicited that inhibition of ERK can reduce TL-induced CEP degeneration by down-regulating the phosphorylation of mTOR. We further detected the protein levels of autophagy markers LC3-II, Beclin-1, and p62 by WB. The results manifested that the levels of LC3-II and Beclin-1 in the TL + si-ERK group were up-regulated and p62 was reduced (all P < .05) (Figure 5F), indicating that suppression of ERK expression can activate autophagy of TL-treated CESCs. Overall, inhibition of ERK expression may alleviate TL-induced CEP degeneration by inhibiting the phosphorylation of key proteins in the MAPK pathway and activating autophagy.OPEN IN VIEWER

In summary, inhibition of the expression of key proteins JNK and ERK in the MAPK pathway alleviated the TL-induced CEP degeneration by inhibiting the phosphorylation of Raptor and mTOR in the mTOR pathway and activating autophagy.