Inhibition of the Mammalian Target of Rapamycin Pathway Stimulates Osteoblast Proliferation and Differentiation Through Autophagy in MC3T3-E1 Cells

Background: OP(Osteoporosis) is a common bone metabolic disorder in the elderly characterized by loss of bone mass and a tendency to fracture. The mammalian target of rapamycin (mTOR) pathway in autophagy plays an indispensable role in maintaining the stability of the intracellular environment and ensuring the normal physiological functions of cells. Methods: In this study, different concentrations(20, 40, 60, 80, 100, 120, 140, 160, 180 and 200nM) of rapamycin were used to act on MC3T3-E1 osteoblasts for different time lengths(6, 12, 24, 36 and 48 hours). CCK8 was used to detect the proliferative activity of cells and screen suitable rapamycin concentration for subsequent experiments. Western blot and real-time quantitative PCR were used to detect the expression changes of phosphorylated mTOR, upstream and downstream mTOR pathway, autophagy and osteogenic differentiation markers. The expression of LC3 was observed by immunouorescence. The differentiation ability of osteoblasts was observed by alizarin red and alkaline phosphatase staining. Results: The results showed that the induction of proliferation activity of osteoblasts from 20 nM to 200 nM presented a parabolic feature. After the action time of 50 μM rapamycin exceeded 12 hours, the proportion of S stage cells was signicantly increased. The results of gene and protein analysis showed that rapamycin signicantly inhibited the phosphorylation of mTOR, and the phosphorylation of the downstream factors of mTOR, 4E-BP1(eIF4E-binding protein 1) and S6K1(p70 ribosomal S6 kinase 1) also decreased. Rapamycin signicantly increased the expression of LC3 II (microtubule associated protein 1 light chain 3-α), signicantly increased the ratio of LC3II/LC3I, and signicantly decreased the expression of p62(sequestosome-1). Rapamycin signicantly induced the expression of ALP(Alkaline phosphatase), Runx2(Runt-related transcription factor 2) and osterix. This study conrmed that rapamycin stimulates the autophagy of osteoblasts by inhibiting mTOR and promotes their proliferation and differentiation, suggesting that mTOR may be a potential therapeutic target for osteoporosis.


Background
Osteoporosis (OP) is a progressive and systemic bone metabolic disease, which is featured by bone mass loss, bone fragility and propensity to fracture (1). The incidence of OP has been increasing yearly due to population aging (2,3). Absent early symptoms and signs are one major feature of OP in clinical practice (4). For this reason, people affected by OP are very likely to suffer osteoporotic fracture, restricted mobility after fracture and other complications. Basic research has shown that the bone marrow mesenchymal stem cells, osteoblasts and osteoclasts are jointly involved in the pathogenesis of OP. The bone is in a dynamic balance of bone formation and absorption. A close coordination between the osteoblasts, osteoclasts, hormones and other factors is required for maintaining bone homeostasis (5). It has been shown that osteoblast apoptosis is enhanced by aging, and reduced bone matrix synthesis resulting from lower osteoblast differentiation is the main mechanism of OP (6).
Autophagy is a highly conservative behavior in eukaryotic cells, which is closely related to cellular homeostasis and stress, damage repair, proliferation and differentiation (7). Autophagy plays an important role in maintaining intracellular homeostasis and normal physiological functions of cells (8).
Among the molecular studies of cellular autophagy, the mammalian target of rapamycin (mTOR) signaling pathway is most intensively discussed. mTOR participates in a variety of biological processes, including cell development, ribosome formation and metabolic regulation (9). mTORC1 induces the phosphorylation of ULK1-ATG13-ATG101-FIP200 complex, leading to its inactivation (10). repamycin activates ULK1 by inhibiting mTORC1, thereby enhancing the activity of the Beclin1-VPS34-ATG14L-p150 complex and triggering autophagy (11). It has been found that the mTORCl activity in osteoclast precursor cells promotes proliferation of osteoblasts, but inhibits their differentiation (12). Other reports have indicated that raptor knockout in osteoblast precursor cells leads to suppressed mTORC1 activity, along with reduced trabecular bones and thinned cortical bones (13).
A growing body of evidence seems to show that autophagy is involved in the regulation of bone metabolism, proliferation, differentiation, metabolism and physiological functions of bone marrow mesenchymal stem cells and osteoblasts (14). ATG7 and the wingless/int1 (Wnt) signaling pathway are involved in the BMP-2-induced early formation of osteoblasts. BMP-2 promotes the expression of ATG7, but LC3, Beclin1 and ATG5 are not affected. After ATG7 and Wnt16 are silenced by small interfering RNA, the BMP-2-mediated differentiation of human skeletal muscle stem cells into osteoblasts is signi cantly inhibited In contrast, the autophagy agonist repamycin dramatically enhances the osteogenic differentiation of hSMSCs, indicating that autophagy promotes early differentiation of osteoblasts (15).
Although autophagy has been proven to regulate osteoblast differentiation, the speci c mechanism remains unclear. The purpose of the present study was to verify whether the mTOR signaling pathway promoted osteoblast differentiation by inducing autophagy.
Analysis of the cell cycle A Cell Cycle Detection kit (KeyGen Biotech Co. Ltd, Nanjing, China) was used to assess the cell cycle.
Following treatment with 50 nM rapamycin for 48 hours, the MC3T3-E1 cells were trypsinized, collected and washed with PBS. Subsequently, 70% cold ethanol was added to x the cells for 2 h at room temperature or overnight at 4°C, and PBS was used to wash away the xing solution. Cells were incubated with 0.4 mL PI containing 0.1 mL RNase A at 37°C for 30 min. Finally, cell cycle distribution was analyzed by measuring the DNA content using a ow cytometer.

Western blot analysis
The protein level were determined by Western blotting. MC3T3-E1 osteoblasts were seeded into 100 mm dishes and cultured in complete α-MEM to 70% con uency, following which the cells were treated with 50 nM rapamycin for 6, 12, 24, 36 and 48 hours. Total proteins were extracted following lysis of the cells in RIPA buffer at 4°C for 1 h. The lysates were centrifuged at 20000 rpm for 30 min, and the protein concentration was determined using a BCA protein quanti cation kit. Equal amounts of protein (30 μg) were loaded into each well of a 7.5-15% SDS-PAGE gel and transferred to PVDF membranes (Millipore Cruz, CA, USA). Adding secondary antibody (goat anti-rabbit IgG-HRP, 1:5000, Santa Cruz) incubated for 2 h. The speci c bands were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scienti c) and imaged with an Alpha Imager HP (ProteinSimple, San Jose, CA, USA). The band density was quanti ed using the ImageJ image processing program (National Institutes of Health, Bethesda, MD, USA. software version 1.5b).
Immuno uorescence staining MC3T3-E1 osteoblasts were seeded in 24-well plates with culture slides (Beyotime Institute of Biotechnology, Shanghai, China) and upon reaching 50% con uency, were treated with 50 nM rapamycin for 12, 24, 36 and 48 hours. The cells were washed twice with PBS, xed with 4% paraformaldehyde at room temperature for 15 min, and permeabilized with 0.5% Triton X-100 for 2 min at room temperature. After blocking for 2 h with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature, the cells were incubated overnight with anti-LC3-II antibody (1:200) at 4°C. After 1 h incubation with Fluorescein (FITC)-conjugated secondary antibody (1:5000) (Santa Cruz), the cells were counterstained with DAPI for 10 min at room temperature. The stained cells were imaged using confocal uorescence microscopy (magni cation, ×200, DS-U3 Nikon Eclipse CI; Nikon Corporation, Tokyo, Japan).

Reverse transcription and real-time PCR
MC3T3-E1 osteoblasts were seeded at a density of 5×10 4 cells/well in 100 mm dishes for 3 days until the cells reached 70% con uence. The cells were treated with 50 nM rapamycin for 6, 12, 24, 36 and 48 hours as described, and total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scienti c), according to the manufacturer's protocol. Total RNA was extracted and quanti ed by scanning spectrophotomer. A total of 1 μg of RNA was used in the reverse transcription reaction using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c) according to the manufacturer's protocols. For gene-speci c primed cDNA synthesis, the reaction was conducted for 60 min at 42°C. Then, for random hexamer primed synthesis, incubate for 5 min at 25°C. RT was terminated by heating at 70°C for 5 min.

Statistical analysis
All statistical tests were performed by SPSS20.0 statistics software (SPSS, Chicago, USA). All experiments were performed three times. Values are expressed as the mean ± standard deviation (SD). Differences between two groups were analyzed by the LSD and Duncan's test. Independent samples t test or one-way analysis of variance was used in comparison between groups. All statistical analysis was performed using SPSS for Windows ver. 19.0 (IBM Corp., Armonk, NY, USA). p<0.05 was considered to indicate a statistically signi cant difference.

Results
Rapamycin stimulates the proliferation activity of MC3T3-E1 osteoblasts The effect of rapamycin (20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 nM) on MC3T3-E1 cell viability was viewed in Figure 1. The results showed that there was no signi cant change in cell proliferation activity when rapamycin was used for 6 h. From the beginning of 12 h, rapamycin above 20 nm signi cantly promoted the proliferation of osteoblasts (p<0.05, p<0.01, respectively). With the increase of rapamycin concentration, the proliferation promoting effect of rapamycin on osteoblasts gradually decreased. The results showed that rapamycin had two-way effect on osteoblasts, which was promoted by low to medium concentration and inhibited by high concentration. According to the above experimental results, 50 nM rapamycin was selected as the subsequent induction condition.

Rapamycin increases the ratio of S-phase in MC3T3-E1 osteoblasts
The cell cycle of each group was determined by ow cytometry. When 50 nM rapamycin was added, compared with the ratio of MC3T3-E1 cell in the control group (75.5% in G1 phase and 7.9% in S-phase), the cell ratio in G1 phase in the rapamycin group was reduced signi cantly ( Rapamycin inhibits the mTOR signaling pathway in MC3T3-E1 osteoblasts PI3K-Akt-mTOR is a classical autophagy signaling pathway, and rapamycin is an inhibitor of mTOR. After the action of 50 nM rapamycin in MC3T3-E1 cells, the expression of mTOR in cells was signi cantly reduced (p<0.01), and the longer the interaction time, the lower the phosphorylation level of mTOR. At the same time, rapamycin also resisted the upstream pathway of mTOR, which showed that the expression of PI3K was signi cantly decreased in the induction groups (p<0.01), and the phosphorylation of Akt was inhibited, and the expression of p-Akt was signi cantly decreased (p<0.05, p<0.01, respectively). These results indicated that rapamycin negatively regulate the phosphorylation of PI3K/Akt/mTOR in MC3T3-E1 cells ( Figure 3A and 3B). After mTOR activation, it mainly regulates the downstream factors of 4EBP1 and S6K1. After 50 nM rapamycin was applied to MC3T3-E1 cells, the phosphorylation level of mTOR in the cells gradually decreased with the extension of the action time, while the phosphorylation levels of its downstream 4EBP1 and S6K1 were signi cantly reduced ( Figure 4A and 4B).

Rapamycin stimulates autophagy in MC3T3-E1 osteoblasts
To analyze the effects of rapamycin on autophagy in MC3T3-E1 osteoblasts, the cells were treated with 50 nM rapamycin, and various autophagy markers LC3-II and p62 were detected. The ratio of LC3-II/LC3-I is frequently analyzed to determine the extent of autophagy. After 6 hours of 50 nM rapamycin treatment, the LC3-II conversion rate of osteoblasts increased steadily, and the expression of LC3-II was signi cantly enhanced. The LC3-II/LC3-I ratio increased signi cantly with the extension of rapamycin treatment time, and p62 showed a signi cant decreasing trend (p<0.05, p<0.01, respectively). In the action time node of rapamycin, only 12 h showed special performance, and rapamycin signi cantly inhibited the formation of LC3-II (p<0.05) ( Figure 5A and 5B). Furthermore, immuno uorescence staining of cells treated with rapamycin was carried out to detect the colocalization of LC3-II, and the results showed that in addition to the decrease in the uorescence intensity of LC3-II at 12 h, the uorescence intensity of LC3-II was signi cantly increased at 24 h after rapamycin treatment, and the number of apoptotic bodies in circular green uorescence was signi cantly increased ( Figure 6A-6E). To further clarify the effects of rapamycin on autophagy, changes in the gene expressions of LC3-II, p62 and mTOR were detected by qRT-PCR. The results showed that the gene expressions of mTOR and p62 were obviously inhibited by rapamycin compared with control (p<0.05, p<0.01, respectively). LC3-II was signi cantly increased except 48 h group (p<0.05, p<0.01, respectively) ( Figure 7). These results demonstrated that rapamycin stimulated autophagy in MC3T3-E1 osteoblasts.

Rapamycin promotes MC3T3-E1 osteoblast differentiation
To detect the function of rapamycin in osteoblast differentiation, western blot assay and real-time quantitative PCR were used to detect the expression of transcription factors related to osteoblast differentiation including ALP, Runx2 and osterix. Treatment with 50 nM rapamycin signi cantly increased ALP activity (p<0.01), except 12 h group. The enhancement of ALP activity was concentration dependent on rapamycin. In addition to 12 h group, rapamycin also promoted the expression of Runx2 and osterix (p<0.05, p<0.01, respectively) ( Figure 8A and 8B). The speci c performance of rapamycin in 12 h was consistent with its effect on the autophagy marker LC3-II. By further gene expression detection, ALP, Runx2 and osterix were expressed in the same direction as protein level, and rapamycin signi cantly induced ALP, Runx2 and osterix expression at the gene level (p<0.05, p<0.01, respectively) ( Figure 9). ALP can be stained with calcium and cobalt to form black deposits in the cells, which can be used to observe the activity of alkaline phosphatase in osteoblasts. The results showed that with the extension of rapamycin treatment time from 6 h to 48 h, the black precipitation of osteoblasts increased and the alkaline phosphatase staining gradually deepened ( Figure 10). Further, alizarin red S staining was used to observe the formation of mineralized nodules in osteoblasts induced by rapamycin. The results showed that after 12 d of rapamycin action, signi cant mineralized nodules could be observed in the cells, and a few calcium salt crystals could be observed at 2 d and 6 d ( Figure 11). These results indicated that rapamycin enhanced osteoblast differentiation.

Discussion
External stimuli (e.g., ischemia, hypoxia, brain damage and convulsion) may induce autophagy, whereby the cells clear way the intracellular damaged organelles and other waste components (16). Autophagic regulation implicates several complex processes, where mTOR, Beclin1, Ca 2+ and PI3K signaling pathways are involved (17). mTORCl activity plays an important role in autophagosome formation and maturation, while repamycin blocks the mTORCl-mediated autophagy (18). After the mTORC1 activity is strengthened, S6K1 is phosphorylated and activated, which further induces the phosphorylation of P70S6 and promotes mRNA translation (19). Meanwhile, migration of ribosomes to the endoplasmic reticulum is accelerated, thereby promoting the adhesion of ribosomes to the endoplasmic reticulum. This further prevents ribosome falling off the endoplasmic reticulum, thereby promoting the maturation of the autophagic membrane (19,20). Given the facts above, autophagy is regulated by the mTORCl signaling pathway. Autophagy is involved in several pathological and physiological processes of human organs (21,22). However, the roles played by autophagy in bone homeostasis and metabolism remain to be further investigated. MC3T3-E1 osteoblasts are usually used to investigate the osteogenic features of bone differentiation and mineralization, which are appropriate for research about the molecular mechanism of osteoblast maturation and extracellular matrix formation (23). In the present study, MC3T3-E1 cells were used to observe the osteogenic features and the induction of autophagy by repamycin in osteoblasts.
Osteoblasts were treated with different concentrations of rapamycin and proliferation of osteoblasts was detected with the cck-8 kit. Cell cycle changes were measured using a cell cycle assay kit. The results showed that with the increase of rapamycin concentration, the proliferation of osteoblasts increased signi cantly, and the reverse inhibition was observed after a certain concentration was exceeded. Changes in cell cycle are related to concentration. The higher the concentration of rapamycin was, the higher the proportion of cells entering S phase from G1 phase, indicating that rapamycin promoted the proliferation of osteoblasts.
The PI3K/Akt/mTOR pathway is an important intracellular signaling pathway regulating autophagy and cell cycle, and it is directly related to cell dormancy, proliferation, carcinogenesis and lifespan (24,25).
PI3K activation induces the phosphorylation and activation of Akt, which is localized at the cytoplasm (26). The signal is transmitted to the downstream via Akt, activating mTOR (27,28). The results showed that repamycin, as an mTOR inhibitor, signi cantly inhibited the phosphorylation of mTOR. Meanwhile, the phosphorylation of the Akt signaling pathway upstream of mTOR was inhibited evidently, while the expression level of Akt itself remained unchanged. The PI3K expression was also downregulated to a great extent. The experiment showed that repamycin not only inhibited the mTOR activity, but also suppressed the activity of Akt.
During the process of autophagic formation, the cytoplasmic material enveloped by the double-sided membrane is called autophagosome (29,30). Autophagosomes bind to lysosomes to degrade the relevant cytoplasmic structures (31). The primary autophagy proteins involved in autophagosome formation are ATG5, beclin-1 and LC3, the early changes of which may in uence the subsequent bone development process (32,33). In recent years, more experiments seem to demonstrate that the osteoblast differentiation is accompanied by upregulated LC3II and SQSTM1/p62, although the interaction between the two is less known (34). The LC3II/LC3I ratio can be used to detect the generation of autophagic ux. After lipidization, LC3 undergoes a transformation from LC3I to LC3II, and the lipidated LC3 is considered as a marker for autophagosome formation (35). The present study proved the repamycin -mediated transformation from LC3I to LC3II at the genetic and protein levels, with a signi cant increase in the LC3II/LC3I ratio. Moreover, the degradation of p62 was increased, indicating that repamycin signi cantly induced the autophagy of osteoblasts. The expression levels of osteogenic markers ALP, Runx2 and osterix were detected to determine osteoblast differentiation (36). Results showed that with the addition of repamycin into the osteoblasts, the expressions of ALP, Runx2 and osterix were increased signi cantly at the genetic and protein levels. Under the action of rapamycin ALP staining obviously deepened, the formation of mineralized nodules, late show that rapamycin targeted by inhibiting mTOR phosphorylation, also inhibit the upstream pathways Akt phosphorylation at the same time, to enhance the osteoblast autophagy level, role in early and late all can promote the osteoblast differentiation, prompting mature osteoblast, strengthen the osteogenetic activity.

Conclusion
Our study demonstrated that rapamycin signi cantly inhibited the phosphorylation of mTOR and Akt, thereby blocking the activation of mTOR downstream pathway 4EBP1 and S6K1, while enhancing the level of autophagy in osteoblasts. The proliferation and differentiation of osteoblasts were also improved. However, the key downstream molecules related to autophagy are not yet identi ed, and other pathways may be involved. Moreover, animal experiments may be required to clarify this issue. Our experiment only demonstrates that autophagy may be correlated with bone metabolic diseases, such as OP. Taken together, it was found that repamycin promoted osteoblast differentiation while inducing osteoblast autophagy, indicating that autophagy may be a potential target for the treatment of OP.
Declarations Figure 1 Effects of rapamycin on MC3T3-E1 cell viability. MC3T3-E1 cells were seeded in 96-well plate and then were exposed to 20, 40, 60, 80, 100, 120, 140, 160 and 200 nM rapamycin for 6, 12, 24, 36, and 48 hours. Cells in the control group were cultured with α-MEM (rapamycin -free) for the same period. The Cell Counting Kit-8 (CCK-8) was used to determine the number of viable cells under different experimental condition. Absorbance was measured at 450 nm in a spectrophotometer. Average optical density (OD) value of cell viability was represented as the mean ± standard deviation. Signi cance analysis of the experimental data for each group was performed using one-way analysis of variance and Tukey's multiple comparisons test (n=8). *p<0.05, **p<0.01 vs. control group.

Figure 2
Effects of rapamycin on MC3T3-E1 cell cycle. Cells were treated with 50 nM rapamycin for 6, 12, 24, 36, and 48 hours as induction groups. Cells in the control group were cultured with α-MEM (rapamycin-free) for the same period. (A) Cell cycle analysis using ow cytometry following PI staining and (B) quanti cation of the cell cycle populations demonstrated that the proportion of cells in G0/G1, S and G2/M phases changed in the rapamycin treatment group compared with that in the control. Values are expressed as the mean ± standard deviation (n=3). *p<0.05, **p<0.01 vs. respective control group.

Figure 3
The relative protein levels of PI3K/Akt/mTOR signaling pathway in MC3T3-E1 cells exposed to rapamycin. Cells were treated with 50 nM rapamycin for 6, 12, 24, 36, and 48 hours. Cells in the control group were cultured with α-MEM (rapamycin-free) for the same period. Cells were harvested with cell lysis buffer. Whole-cell extracts from MC3T3-E1 cells were separated on SDS-PAGE for Western blot analysis using antibody speci c to phosphorylation of mammalian target of rapamycin (p-mTOR), protein kinase B (Akt), p-Akt and phosphatidylinositol 3'-kinase (PI3K). Data shown are the mean ± SD (n=3). *p<0.05, **p<0.01 vs. respective control group.

Figure 4
The relative protein levels of mTOR signaling pathway in MC3T3-E1 cells exposed to rapamycin. Cells were treated with 50 nM rapamycin for 6, 12, 24, 36, and 48 hours. Cells in the control group were cultured The relative protein levels of autophagy in MC3T3-E1 cells exposed to rapamycin. Cells were treated with 50 nM rapamycin for 6, 12, 24, 36, and 48 hours as induction groups. Cells in the control group were cultured with α-MEM (rapamycin-free) for the same period. Cells were harvested with cell lysis buffer. Whole-cell extracts from MC3T3-E1 cells were separated on SDS-PAGE for Western blot analysis using  The relative RNA abundance of autophagy signaling pathway in MC3T3-E1 cells exposed to rapamycin.
Cells were treated with 50 nM rapamycin for 6, 12, 24, 36, and 48 hours as induction groups. Cells in the control group were cultured with α-MEM (rapamycin-free) for the same period. Quantitative PCR was performed with an SYBR Green PCR Master Mix. Average relative RNA abundance of immunoglobulim heavy chain LC3-II, p62 and mTOR are represented as mean ± SD (n=3). *p<0.05, **p<0.01 vs. respective control group.

Figure 8
The relative protein levels of transcription factors related to osteoblast differentiation in MC3T3-E1 cells exposed to rapamycin. Cells were treated with 50 nM rapamycin for 6, 12, 24, 36, and 48 hours as induction groups. Cells in the control group were cultured with α-MEM (rapamycin-free) for the same period. Cells were harvested with cell lysis buffer. Whole-cell extracts from MC3T3-E1 cells were separated on SDS-PAGE for Western blot analysis using antibody speci c to Runt-related transcription factor 2 (Runx2), osterix and alkaline phosphatase (ALP). Data shown are the mean ± SD (n=3). *p<0.05, **p<0.01 vs. respective control group.

Figure 9
The relative RNA abundance of transcription factors related to osteoblast differentiation in MC3T3-E1 cells exposed to rapamycin. Cells were treated with 50 nM rapamycin for 6, 12, 24, 36, and 48 hours as induction groups. Cells in the control group were cultured with α-MEM (rapamycin-free) for the same period. Quantitative PCR was performed with an SYBR Green PCR Master Mix. Average relative RNA abundance of immunoglobulim heavy chain ALP, Runx2 and osterix are represented as mean ± SD (n=3). *p<0.05, **p<0.01 vs. respective control group.