Mechanical Stress-Induced IGF-1 Facilitates col-I and col-III Synthesis via the IGF-1R/AKT/mTORC1 Signaling Pathway

Mechanical stress promotes human ligamentum flavum cells (LFCs) to synthesize multitype collagens, leading to ligamentum flavum hypertrophy (LFH). However, the mechanism of mechanical stress in the formation of collagen remains unclear. Therefore, we investigated the relationship between mechanical stress and collagen synthesis in the present study. First, LFCs were isolated from 9 patients and cultured with or without mechanical stress exposure for different times. IGF-1, collagen I (col-I), and collagen III (col-III) protein and mRNA levels were then detected via ELISA and qPCR, respectively. Moreover, the activation of pIGF-1R, pAKT, and pS6 was examined by Western blot analysis. To further explore the underlying mechanism, an IGF-1 neutralizing antibody, NVP-AEW541, and rapamycin were used. IGF-1, col-I, and col-III were significantly increased in stressed LFCs compared to nonstressed LFCs. In addition, the activation of pIGF-1R, pAKT, and pS6 was obviously enhanced in stressed LFCs. Interestingly, col-I protein, col-I mRNA, col-III protein, col-III mRNA, and IGF-1 protein, but not IGF-1 mRNA, were inhibited by IGF-1 neutralizing antibody. In addition, col-I and col-III protein and mRNA, but not IGF-1, were inhibited by both NVP-AEW541 and rapamycin. Moreover, the activation of pIGF-1R, pAKT, and pS6 was reduced by the IGF-1 neutralizing antibody and NVP-AEW541, and the activation of pS6 was reduced by rapamycin. In summary, these results suggested that mechanical stress promotes LFCs to produce IGF-1, which facilitates col-I and col-III synthesis via the IGF-1R/AKT/mTORC1 signaling pathway.


Introduction
Currently, an increasing number of elderly individuals have lumbar spinal stenosis (LSS) [1,2]. The clinical symptoms of LSS include lower limb numbness with pain, low back pain, and claudication [3]. LSS causes tremendous discomfort for patients, and LSS is often caused by ligamentum flavum hypertrophy (LFH) [4,5]. Previous studies [6][7][8][9] have shown that mechanical stress promotes collagen I (col-I) and collagen III (col-III) synthesis which contributes to LFH. However, the exact mechanisms remain unclear.
In the present study, we hypothesized that mechanical stress plays a pivotal role in IGF-1 synthesis. In addition, IGF-1 may promote col-I and col-III synthesis by the IGF-1R/AKT/mTORC1 signaling pathway. To test these hypotheses, col-I and col-III as the important indicator of LFH were detected, and related marker activation of the IGF-1R/AKT/ mTORC1 signaling pathway was examined. In addition, the relationship of mechanical stress and IGF-1 in LFH as well as the potential mechanism involved was investigated.

Materials and Methods
2.1. LFC Cultivation and Identification. First, nonthickened ligamentum flavum (LF) samples were aseptically obtained from 9 lumbar surgery patients (5 males and 4 females with an average age of 47.2 years). The LF samples were washed with physiological saline 3 times before being minced into 0.5 mm 3 pieces. The LF samples were then digested with 0.2% collagenase-I for 1.5 h at 37°C and washed 2 times with PBS. The LF samples were centrifuged 3 times at 1000 r/min for 5 min before being placed in cell culture plates with DMEM containing 20% fetal bovine serum (FBS). Subsequently, The LF samples were incubated, and the medium was changed every 3 days. Approximately one week later, LFCs migrated out from the LF samples. When the LFCs reached 80% confluence, they were passaged 1 : 2. Moreover, some of the LFCs were cryopreserved in media (10%DMSO + 20%FBS + 70%DMEM) at -80°C. LFC morphology was inspected, and the expression of vimentin and col-I in LFCs was detected by immunostaining [21].

Mechanical Stress Application.
Experiments were performed with LFCs from each individual patient. The LFCs were grouped into experimental and control groups, and they were cultured in BioFlex I 6-well plates at 1 × 10 5 cells per well. After LFCs reached 80% confluence, they were subjected to serum starvation (DMEM with 0.2% FBS) for 12 h. The experimental groups were subjected to cycles of relaxation for 10 s and 20% elongation for 10 s by a tension system (FX5K, Flexcell International Corporation, USA) [22][23][24][25] for 6 h, 12 h, and 24 h. Control groups were cultured in the same environment without mechanical stress.
2.3. IGF-1 Neutralizing Antibody Treatment. LFCs were grouped into the following 3 groups: nonstress group, stress group, and stress+IGF-1 neutralizing antibody (10 μg/ml, Abcam, Cambridge, UK) group. LFCs in the stress group and the stress+IGF-1 neutralizing antibody group were subjected to cycles of relaxation for 10 s and 20% elongation for 10 s by the tension system for 24 h, and LFCs in the nonstress group were not subjected to mechanical stress. IGF-1, col-I and col-III protein, and mRNA levels were detected by ELISA and RT-qPCR, respectively, in each group. In addition, the activation of pIGF-1R, pAKT, and pS6 in each group was evaluated by Western blot analysis.
2.4. NVP-AEW541 Treatment. LFCs were grouped into the following 3 groups: nonstress group, stress group, and stress+100 ng/ml NVP-AEW541 (a specific inhibitor of IGF-1R, dissolved in DMSO, MedChem Express, Monmouth Junction, NJ) group. LFCs in the stress group and the stress+100 ng/ml NVP-AEW541 group were subjected to cycles of relaxation for 10 s and 20% elongation for 10 s by the tension system for 24 h, and LFCs in the nonstress group were not subjected to mechanical stress. IGF-1, col-I and col-III protein, and mRNA levels were detected by ELISA and RT-qPCR, respectively, in each group. Moreover, the activation of pIGF-1R, pAKT, and pS6 in each group was evaluated by Western blot analysis.
2.5. Rapamycin Treatment. LFCs were grouped into the following 3 groups: nonstress group, stress group, and stress+10 ng/ml rapamycin (a specific inhibitor of mTORC1, dissolved in DMSO, Alexis Biochemicals, Lausen, Switzerland) group. LFCs in the stress group and the stress+10 ng/ ml rapamycin group were subjected to cycles of relaxation for 10 s and 20% elongation for 10 s by the tension system for 24 h, and LFCs in the nonstress group were not subjected to mechanical stress. IGF-1, col-I and col-III protein, and mRNA levels were detected by ELISA and RT-qPCR, respectively, in each group. Moreover, the activation of pS6 in each group was evaluated by Western blot analysis.
2.6. RT-qPCR Analysis. IGF-1, col-I, and col-III mRNA was measured by RT-qPCR in each group. First, we extracted total RNA from LFCs and detected its concentration and purity. Reverse transcription was then performed followed by qPCR. The primer sequences used in the present study are listed in Table 1 [21]. Sangon Biotech (Sangon Biotech, China) synthesized all primers in the study. All assays in the study were performed in triplicate. The samples were normalized to GAPDH and analyzed by the 2 -ΔΔCq method [26].
2.7. Enzyme-Linked Immunosorbent Assay. Culture supernatants from LFCs in each group were collected. To remove insoluble impurities and cell debris, the supernatants were centrifuged at 1000 g at 4°C for 20 min. The cleared supernatants were immediately used to measure IGF-1, col-I, and col-III protein levels by a Human IGF-1 ELISA Kit (Elabscience Biotechnology, China), Collagen I ELISA Kit (Elabscience Biotechnology, China), and Collagen III ELISA Kit (Elabscience Biotechnology), respectively.

Western Blot Assays.
LFCs from each group were lysed on ice in lysis buffer, and the lysates were then added to Laemmli buffer at 100°C for 10 min. The LFC lysates were separated by electrophoresis, and the proteins were then transferred to nitrocellulose membranes. The membranes were blocked with TBS containing 5% nonfat milk for 2 h at 25°C. Subsequently, the membranes were incubated with primary antibodies for 12 h at 4°C followed by incubation with secondary antibodies for 1.5 h at 25°C. Finally, a chemiluminescence kit (Beyotime, China) was used to visualize the nitrocellulose membranes.

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Stem Cells International 2.9. Statistical Analyses. Data were statistically analyzed and graphed using GraphPad Prism 5.01 (GraphPad Software Inc., San Diego, CA, USA). Protein and mRNA changes with or without mechanical stress at different times were analyzed by one-way ANOVA, and Tukey' s honestly significant difference was used as the post hoc method. The remaining data were analyzed by Student's t-test. The results were considered significant when P < 0:05, and the data are presented as the mean ± SD.

Identification and Morphology of LFCs with or without
Mechanical Stress. Immunofluorescence staining showed that LFCs expressed high levels of col-I and vimentin (Figure 1), which indicated that highly purified LFCs were cultured. Without mechanical stress, most LFCs were polygonal ( Figure 2(a)). Under mechanical stress, LFCs became fusiform and arranged along the direction of stress ( Figure 2(b)).
First, we isolated primary LFCs from 9 patients who underwent lumbar spinal surgery. LFCs are fibrous connective tissue stem cells. According to the study conducted by Zhong and Chen [39], LFCs can be identified by detecting col-I and vimentin expression. Therefore, in the present     Figure 4: IGF-1 neutralizing antibody treatment. Mechanical stress in the nonstress group, the stress group, and the stress+10 μg/ml IGF-1 neutralizing antibody group. The IGF-1 neutralizing antibody reduced the mRNA levels of col-I (f) and col-III (g), but not IGF-1 (e). The IGF-1 neutralizing antibody also reduced the protein expression of IGF-1 (h), col-I (i), and col-III (j). In addition, the activation of pIGF-1R (a, b), pAKT (a, c), and pS6 (a, d) was reduced by the IGF-1 neutralizing antibody. Columns represent the mean ± SD of 3 samples, and each experiment was performed in triplicate. LFCs: ligamentum flavum cells; IGF-1: insulin-like growth factor 1; col-I: collagen I; col-III: collagen III. " * " represents P < 0:05; "ns" indicates P > 0:05.

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Stem Cells International  Figure 5: NVP-AEW541 treatment. Mechanical stress in the nonstress group, the stress group, and the stress+100 ng/ml NVP-AEW541 group. NVP-AEW541 reduced the mRNA levels of col-I (f) and col-III (g), but not IGF-1 (e). col-I (i) and col-III (j) protein levels, but not IGF-1 (h) protein levels, were attenuated by NVP-AEW541. In addition, NVP-AEW541 reduced the activation of pIGF-1R (a, b), pAKT (a, c), and pS6 (a, d). Columns represent the mean ± SD of 3 samples, and each experiment was performed in triplicate. LFCs: ligamentum flavum cells; IGF-1: insulin-like growth factor 1; col-I: collagen I; col-III: collagen III. " * " represents P < 0:05; "ns" indicates P > 0:05. 7 Stem Cells International study, LFC purity was examined by col-I and vimentin expression. Immunofluorescence results showed that the LFCs were of high purity (Figure 1). In addition, LFC viability was evaluated by the MTT assay (Solarbio, China), which demonstrated that there were no changes in LFC viability in each group.
LFCs were also subjected to cyclic mechanical stress at different times. Compared to nonstressed cells, cyclic mechanical stress promoted the synthesis of IGF-1 in LFCs in a time-dependent manner, which eventually led to col-I/ col-III accumulation via the IGF-1R/AKT/mTORC1 signaling pathway. To understand the molecular mechanism involved, we used an IGF-1 neutralizing antibody. Compared to the nonstress group, IGF-1, col-I, and col-III protein and mRNA levels were increased in the stress group. In addition, col-I and col-III protein and mRNA levels were significantly reduced in the stress+IGF-1 neutralizing antibody group compared to the stress group. Interestingly,  Figure 6: Rapamycin treatment. Mechanical stress in the nonstress group, the stress group, and the stress+rapamycin group. Rapamycin reduced the mRNA levels of col-I (d) and col-III (e), but not IGF-1 (c). Rapamycin reduced the protein levels of col-I (g) and col-III (h), but not IGF-1 (f). In addition, rapamycin reduced the activation of pS6 (a, b). Columns represent the mean ± SD of 3 samples, and each experiment was performed in triplicate. LFCs: ligamentum flavum cells; IGF-1: insulin-like growth factor 1; col-I: collagen I; col-III: collagen III. " * " represents P < 0:05; "ns" indicates P > 0:05.

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Stem Cells International IGF-1 protein expression, but not mRNA expression, was reduced in the stress+IGF-1 neutralizing antibody group compared to the stress group. Correspondingly, the activation of pIGF-1R, pAKT, and pS6 was decreased in the stress+IGF-1 neutralizing antibody group compared to the stress group. For further investigation, NVP-AEW541 (a specific inhibitor of IGF-1R) and rapamycin (a specific inhibitor of mTORC1) were used in the present study. Although 100 ng/ml NVP-AEW541 and 10 ng/ml rapamycin almost completely blocked the IGF-1R/AKT/mTORC1 signaling pathway, col-I and col-III protein and mRNA levels were only partially reduced. col-I and col-III protein and mRNA levels were still higher in both groups compared to the nonstress group. Moreover, neither NVP-AEW541 nor rapamycin reversed IGF-1 expression, which was induced by mechanical stress. Based on the above findings, we hypothesized that mechanical stress may promote col-I and col-III production via other signaling pathways, and the potential mechanism involved requires further study. In addition, due to the lack of an animal model of LFH, only cytological experiments were performed in the present study. Thus, it is necessary to build an effective animal model for further research.

Conclusion
In summary, the present study showed that mechanical stress upregulated IGF-1, col-I, and col-III protein and mRNA production. The IGF-1 neutralizing antibody, NVP-AEW541, and rapamycin blocked the IGF-1R/AKT/ mTORC1 signaling pathway and reduced col-I and col-III production in LFCs. These findings demonstrated that cyclic mechanical stress promotes LFCs to secrete IGF-1, which induces col-I and col-III synthesis via the IGF-1R/AKT/ mTORC1 signaling pathway (Figure 7). These results provide a new understanding of LFH and may facilitate the development of novel methods to treat LSS.

Data Availability
Data are available on request (detail contact information: nfzzm@163.com).

Conflicts of Interest
The authors in the study declare that they have no conflicts of interest.

Authors' Contributions
Bin Yan, Canjun Zeng, and Yuhui Chen contributed equally to this work.

Acknowledgments
The National Natural Science Foundation of China (No. 31801012) and Science and Technology Program of Guangzhou, China (201804010390), supported this study.