Role of autophagy on bone marrow mesenchymal stem‑cell proliferation and differentiation into neurons

  • Authors:
    • Bo Li
    • Ping Duan
    • Caifang Li
    • Ying Jing
    • Xuefei Han
    • Wenhai Yan
    • Ying Xing
  • View Affiliations

  • Published online on: December 10, 2015     https://doi.org/10.3892/mmr.2015.4673
  • Pages: 1413-1419
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Abstract

The purpose of the present study was to investigate the role of autophagy on rat bone marrow mesenchymal stem cell (BMSC) proliferation, apoptosis and differentiation into neurons. After treatment with rapamycin, 3‑methyladenine (3‑MA) or chloroquine, the cell cycle, apoptosis, expression of neuron‑specific enolase (NSE) and the mean fluorescence intensity (MFI) of Notch1 in BMSCs were examined by flow cytometry. The expression of microtubule‑associated protein 2 (MAP2), Notch1 and Hes1 was investigated by western blot analysis. The results showed that after induction of autophagy using rapamycin, the proliferation of BMSCs was inhibited. Furthermore, the S‑phase population was significantly decreased compared to that in the control group (P<0.05). In addition, the percentage of NSE‑positive cells and the expression of MAP2 were significantly increased compared to those in the control group (P<0.05). The MFI of Notch1 was markedly upregulated compared to that in the control group (P<0.05). When autophagy was inhibited by 3‑MA or chloroquine, the percentage of apoptotic cells and NSE‑positive cells as well as the expression of MAP2 were markedly reduced compared to those in the control group (P<0.05). Furthermore, western blot analysis showed that Notch1 and Hes1 were decreased in the rapamycin‑treated group, while they were not affected by 3‑MA or chloroquine. The present study indicated that induction of autophagy in BMSCs decreased their S‑phase population, promoted their differentiation into neurons and promoted the expression of NSE and MAP2. The mechanisms underlying this process may be linked to the regulation of autophagy‑induced inhibition of the Notch1 signaling pathway.

Introduction

Autophagy is a conserved and important metabolic pathway in eukaryotic cells. Under starvation or stress conditions, harmful and unnecessary intracellular macromolecules are degraded and recycled through autophagy, which thereby promotes cell survival and growth (1). In populations of undifferentiated cells as well as cells undergoing self-renewal or differentiation into mature cells, the cells exhibit protein renewal and organelle degradation (2). Autophagy, which is stimulated by the macroenvironment or cytokines, has an important role in remodeling stem cells, thus regulating cell differentiation and self-renewal. However, the role of autophagy in bone marrow-derived mesenchymal stem cell (BMSC) proliferation and differentiation has remained elusive.

Notch1 signaling is a regulator which is widely distributed in various organs of developing embryos (3), a variety of adult organs as well as undifferentiated cells with the ability to proliferate, and which has important roles in the development, proliferation and differentiation of various cell types (46). Notch1 signaling has been shown to have important roles in BMSC proliferation and differentiation, where it exerts an activating and inhibitory function, respectively (7). Furthermore, induction of autophagy is associated with the Notch1 signaling pathway. In order to investigate the role of autophagy in BMSC proliferation and differentiation, as well as the underlying molecular mechanisms, the present study used autophagy inducer rapamycin and autophagy inhibitors 3-methyladenine (3-MA) and chloroquine, to modulate autophagic activity in BMSCs. The effects of the modified autophagic activity on BMSC proliferation, apoptosis and differentiation into neurons were examined, and the role of the Notch1 signaling pathway in this process was investigated.

Materials and methods

Animals

Adult male Sprague-Dawley rats (n=30; age, four weeks, weight, 160–200 g) were obtained from the Animal Center of Zhengzhou University (Zhengzhou, China) and housed in a specific pathogen-free room. Animal procedures were approved by the Ethics Committee of Zhengzhou University (Zhengzhou, China) and performed according to the 'Guidance On the Care and Use of Laboratory Animals' (People's Republic of China National Science and Technology Committee; 2006). BCMCs were obtained three times from 10 rats.

Reagents

The reagents used in the present study were as follows: 0.25% Trypsin-EDTA (1X), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) (all from Gibco; Thermo Fisher Scientific, Waltham, MA, USA), radioimmunoprecipitation assay (RIPA) lysis buffer, phenylmethanesulfonyl fluoride (PMSF), bicinchoninic acid (BCA) protein assay kit (cat. no. P0010s), and Electro-Chemi-Luminescence Plus kit (cat. no. P0018; all from Beyotime Institute of Biotechnology, Haimen, China), Tris, glycine and SDS (all from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), Temed (Shanghai Huashuo Fine Chemical Co., Ltd., Shanghai, China), ammonium persulfate (Tianjin Kemiou Chemical Reagent Co., Ltd., Tianjin, China), rapamycin, 3-MA, chloroquine and dimethylsulfoxide (DMSO) (all from Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-Notch1, rabbit polyclonal anti-Hes1, rabbit monoclonal anti-P62, and rabbit polyclonal anti-microtubule-associated protein 2 (MAP2) antibodies (All from Abcam, Cambridge, MA, USA; cat. nos. ab33932, ab49170, ab109012, and ab32454; 1:1,000), rabbit-anti polyclonal microtubule-associated protein 1 light chain 3B (LC3B), and rabbit-anti polyclonal β-actin antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA; cat. nos. 3868 and 4967; 1:1,000), and horseradish peroxidase-labeled goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Inc.; cat. no. SC-2004; 1:2,000).

Isolation, purification and induction of BMSCs

Following sacrificing the rats by cervical dislocation, bone marrow cells were collected by repeatedly flushing both femurs and tibias with culture medium using a 5-ml syringe under the sterile/cell culture hood. Cells at 1×106/ml were seeded into culture dishes (75 cm2) and cultured in a humidified incubator containing 5% CO2 at 37°C, and the medium was changed every 3–4 days. 6–7 days later, cells were re-seeded at the ratio of 1:2.

Cells in the third passage were randomly divided into four groups: The control group and three groups treated with rapamycin (10 mmol/l), 3-MA (3 mmol/l) and chloroquine (25 mmol/l), respectively, for 12 h. Experiments were performed in triplicate. For the experiments on cell differentiation, cells were divided into the same groups as mentioned above, but cultured with induction medium [low-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 2% DMSO and 200 µmol/l butylated Hydroxyanisole (BHA; Sigma-Aldrich)] for 12 h (8).

Flow cytometry
Cell cycle analysis

After trypsinization and washing in phosphate-buffered saline (PBS) twice, cells were re-suspended with 200 µl cold PBS and fixed with 800 µl chilled ethanol, which was added drop wise with agitation. The suspensions were stored at −20°C overnight, followed by centrifugation (179 × g, 5 min, room temperature), re-suspension of the cell pellet in propidium iodide (PI; BD Biosciences, Erembodegem, Belgium)/RNase staining buffer (200 µl; BD Biosciences, Franklin Lakes, NJ, USA) and incubation in the dark for 15 min at room temperature. The cell cycle was then analyzed by flow cytometry.

Assessment of apoptosis

Cell treatments were conducted according to the protocol of the fluorescein isothiocyanate (FITC)-Annexin V Apoptosis Detection kit (BD Biosciences; cat. no. 556547). After trypsinization and washing in PBS twice, cells were re-suspended in 100 µl 1X Annexin V binding buffer, 5 µl FITC-Annexin V and 5 µl PI (all from BD Biosciences), and incubated in the dark for 15 min after gentle mixing. Following addition of 200 µl 1X Annexin V binding buffer, apoptosis was analyzed by flow cytometry (BD FACSCanto II; BD Biosciences) within 1 h.

Analysis of neuron-specific markers neuron-specific enolase (NSE) and Notch1

Cells were fixed and permeabilized using the BD cytofix/cytoperm kit (cat. no. 554714; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. In brief, 500 µl Fixation/Permeabilization solution was added and cells were incubated in the dark for 20 min at room temperature. After centrifugation (179 × g, 5 min, room temperature), cell pellets were re-suspended in 2 ml BD Perm/wash buffer and incubated in the dark for 10 min at room temperature. Subsequently, phycoerythrin (PE)-conjugated mouse IgG anti-Notch1 antibodies (1 µl; BD Biosciences; cat. no. 552768; 1:100) or rabbit polyclonal anti-NSE (Abcam; cat. no. ab53025; 1:500) were added, followed by incubation for 30 min at room temperature in the dark or 2 h on ice in the dark, respectively. After the cells were washed using BD Perm/wash buffer, the cells incubated with anti-Notch1 antibodies were re-suspended in 300 µl PBS containing 1% paraformaldehyde and stored at 4°C, whereas those incubated with anti-NSE antibodies were treated with PE-conjugated rabbit IgG antibodies (BD Biosciences; cat. no. 558416; 1:1,000) for 1 h on ice in the dark. The cells were then washed and re-suspended as above. The ratio of NSE-positive cells and the mean fluorescence intensity (MFI) of Notch1 were analyzed using flow cytometry (BD FACSCanto II) with a Data-Interpolating Vibrational Analysis software version 5.0 (BD Biosciences) for data acquisition and analysis.

Western blot analysis

Following cell lysis and protein extraction using RIPA lysis buffer, the protein concentration was measured using a BCA assay according to the instructions of the BCA kit. Protein in loading buffer (40 µg) was resolved by 10% sodium dodecyl polyacrylamide electrophoresis (using 80-V constant-voltage electrophoresis until the bromophenol blue entered the gel, followed by 120-V constant-voltage electrophoresis for 90 min; Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) and transferred onto a polyvinylidene difluoride membrane (250 mA for 1 h; Beijing Dingguo Changsheng Biotechnology Co., Ltd.). After blocking with 5% skimmed milk for 1 h at room temperature, membranes were incubated with rabbit-anti LC3B, rabbit-anti MAP2, rabbit-anti P62, rabbit anti-Notch1, rabbit anti-Hes1 (1:1,000) or β-actin antibodies (1:2,000) overnight at 4°C. After washing with Tris-buffered saline containing Tween 20 three times, membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2,000) for 1 h at room temperature. The membranes were further washed three times for 5 min. Bands were detected using the Electro-Chemi-Luminescence Plus kit. Grey value analysis was performed using Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). β-actin was used as a loading control, and the expression levels of the proteins were normalized to β-actin.

Statistical analysis

Values are expressed as the mean ± standard deviation. Statistical analysis was performed using the Student's t-test with SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a significant difference between values.

Results

Pharmacological modulation of autophagic activity in BMSCs

Treatment with autophagy inducer rapamycin for 12 h promoted the expression of LC3B-II and reduced P62 expression in BMSCs, while autophagy inhibitors 3-MA and chloroquine produced opposite effects, except for a slight enhancement of LC3B-II expression following chloroquine treatment (Fig. 1). These results indicated that rapamycin was able to enhance autophagy, while 3-MA and chloroquine inhibited autophagy in BMSCs.

Autophagy inhibits the cell cycle of BMSCs

Cell cycle analysis BMSCs showed that induction of autophagy by rapamycin inhibited the cell proliferation, as indicated by a reduced S-phase cell population (11.04±0.86%) when compared to that in the control group (14.18±0.76%; P<0.05) (Fig. 2). Conversely, treatment with 3-MA and chloroquine marginally increased the S-phase cell population to 15.13±0.17% and 15.56±0.29%, respectively; however, differences from the control group were not statistically significant (Fig. 2).

Autophagy promotes BMSC apoptosis

Flow cytometric analysis showed that autophagy inducer rapamycin slightly enhanced the apoptotic rate of BMSCs (5.8±0.42%), which was, however, not significantly different from that of the control group (4.6±0.55%). Following treatment with autophagy inhibitors 3-MA or chloroquine, the apoptotic rate of BMSCs (1.5±0.1 and 1.23±0.25, respectively) was significantly reduced when compared to that in the control group (P<0.05) (Fig. 2). These results indicated that autophagy promotes apoptosis, while inhibition of autophagy prevents apoptosis of BMSCs.

Autophagy enhances the differentiation of BMSCs into neurons

BMSCs at passage 3 were spindle-shaped and had a fibroblast-like morphology. Following incubation in induction medium (low-glucose DMEM with 2% DMSO and 200 µmol/l BHA) for 12 h, cells exhibited neurite extensions and their shape was changed to resemble neurons. Flow-cytometric analysis showed that rapamycin-induced autophagy promoted BMSC differentiation into neurons, as demonstrated by a significantly increased number of NSE-positive cells (78.2±2.6) compared to that in the control group (36.7±2.8; P<0.05) (Fig. 3A and B). Conversely, treatment with autophagy inhibitors 3-MA or chloroquine reduced the percentage of NSE-positive cells (32.8±3.1 and 29.9±1.56, respectively) compared that in the control group (P<0.05) (Fig. 3A and B). Furthermore, western blot analysis showed that upon differentiation into neurons, the expression of microtubule-associated protein 2 (MAP2) was increased in the rapamycin-treated and reduced in the 3-MA- or chloroquine-treated groups compared to that in the control group (P<0.05 for all) (Fig. 3C and D).

The effect of autophagy on Notch1 signaling pathway in BMSCs

To investigate the role of the Notch1 signaling pathway in the observed autophagy-mediated enhancement of neuronal differentiation of BMSCs, flow cytometry was used to detect Notch1 MFI after modulation of autophagy. The results showed that the Notch1 MFI was significantly reduced after treatment with rapamycin (410±19), when compared to that in the control group (552±23; P<0.05) (Fig. 4A and B). However, treatment with 3-MA or chloroquine did not change the MFI of Notch1 (560±39 and 570±30, respectively) compared to that in the control group. Furthermore, western blot analysis showed that treatment with autophagy inducer rapamycin reduced the expression of Notch1 and Hes1, whereas 3-MA and chloroquine did not affected the expression of Notch1, but both increased Hes1 expression levels (Fig. 4C and D).

Discussion

Transdifferentiation of BMSCs into neurons provides a novel approach for the treatment of neurological diseases by cell transplantation (9,10). However, in vitro, the efficacy of BMSC proliferation and differentiation is low and the function of differentiated mature cells is also limited; furthermore, the underlying mechanisms of these processes have largely remained elusive (1113). Autophagy is a process during which cells digest their own long-lived proteins and organelles under conditions of nutritional starvation and stress (1416). Autophagy has an important role in cell proliferation, differentiation and apoptosis (1720), and can rapidly and efficiently degrade transcription factors, enzymes, adhesion molecules and certain secreted proteins (21,22), which are important for stem-cell proliferation and differentiation (2325). Further studies focusing on autophagy in BMSCs may reveal the underlying mechanisms of proliferation and differentiation, and thereby provide potential strategies for clinical treatment.

The LC3 protein is localized in the membrane surface of autophagosomes. Upon the induction of autophagy, LC3-I binds to phosphatidylethanolamine on the membrane surface of the autophagosome and is then modulated by autophagy-related 7 (Atg7) to form LC3-II. As the levels of LC3-II are positively correlated with the number of autophagic vacuoles, it is considered to be a marker for autophagy (26). P62 is able to bind with LC3 and targeting proteins, which are then degraded in lysosomes. P62 is extensively degraded when the activity of autophagy is increased, while it accumulates in the cytoplasm when autophagy is attenuated, suggesting that P62 levels reflect the level of autophagic activity (27). Mammalian target of rapamycin (mTOR) and the phosphoinositide-3 kinase (PI3K)/AKT pathway are the most important signaling pathways involved in the regulation of autophagy (28). Rapamycin can enhance autophagic activity by inhibiting the mTOR signaling pathway (28), which explains for the result of the present study that rapamycin treatment enhanced LC3-II levels and reduced P62 levels in BMSCs. 3-MA inhibits autophagy by decreasing the activity of type III PI3K signaling (29). Thus, treatment of BMSCs with 3-MA reduced the expression of LC3-II and enhanced P62 in the present study. Chloroquine is able to inhibit autophagy at the late stages by preventing the fusion of autophagosomes and lysosomes (30). Thus, in the present study, treatment with chloroquine slightly increased LC3-II, while it markedly enhanced the expression of P62 in BMSCs.

Deficiency of Atg7 in hematopoietic stem cells has been reported to promote the amplification of bone marrow precursor cells, thus inducing invasive bone marrow hyperplasia and presenting the phenotype of acute myeloid leukemia (25). Autophagy has been suggested to occur in BMSCs (31,32), and is enhanced under stress conditions, such as hypoxia (33). However, the role of autophagy in BMSC apoptosis has remained to be elucidated. Autophagy has been shown to exert cytoprotective effects under stress conditions (34,35). Hypoxia and starvation are known to promote autophagy and decrease apoptosis in BMSC, while 3-MA treatment increases BMSC apoptosis (36). Ugland et al (37) showed that autophagy can be activated via the cyclic adenosine monophosphate (cAMP) signaling pathway in BMSCs, and that the activated cAMP is able to inhibit cell proliferation though modulation of extracellular signal-regulated kinase-mediated activation of cyclin E, thus recruiting autophagy-associated protein Beclin1 to surround the nucleus, which resulted in the formation of autophagic bodies.

In the present study, rapamycin was used to induce autophagy in BMSCs, which was demonstrated to decrease the S-phase population of the cell cycle and promote apoptosis, while treatment with 3-MA and chloroquine exerted the opposite effects, suggesting that activation of autophagy inhibits BMSC proliferation. Autophagy may be a double-edged sword, which can have pro-survival effects under stress conditions to promote cell growth and proliferation, while inhibiting cell proliferation and promoting apoptosis upon excessive activation by degrading proteins and organelles essential for cell proliferation (38).

The present study revealed that rapamycin-induced autophagy enhanced the expression of neuron-specific markers NSE and MAP2. These findings suggested that autophagy is involved in BMSC differentiation. Zeng and Zhou (2) found that autophagy is enhanced during the process of retinoic acid-induced differentiation of N2a neuroblastoma cells. Furthermore, the differentiation ability was inhibited by treatment with autophagy inhibitors 3-MA and LY294002 and by RNA interference-mediated suppression of Beclin1. In addition, the mTOR pathway was shown to be inhibited during N2a neuroblastoma cell differentiation, and treatment with mTOR inhibitor rapamycin promoted neurite extension, increased the cell size and enhanced the expression of neuron-specific markers (1). Furthermore, deficiency of Atg7 and Atg5 as well as treatment with 3-MA has been shown to inhibit BMSC differentiation into adipocytes, suggesting that autophagy has an important role in BMSC differentiation into adipocytes (39). Li et al (40) have shown that autophagy is activated during the process of BMSC differentiation into neurons, and that treatment with rapamycin increased the expression levels of Tau and MAP2, while 3-MA had the opposite effect.

The present study provided evidence that the degradation of 'stemness'-associated proteins and the production of neuron-specific proteins is part of the process of BMSC differentiation into neurons. Induction of autophagy was indicated to accelerate this process and thus represents an approach for stimulating cell differentiation, increasing the expression of neuron-specific markers, and promoting BMSC differentiation into neurons.

Notch, a regulatory factor and an important signaling pathway controlling cell fate, has an important role in growth, development, proliferation, differentiation and apoptosis of various cell types (4143). A previous study by our group demonstrated that the expression of Notch1 and Jag1 genes and their downstream targeted genes PS1 and Hes1 were significantly reduced during the process of human mesenchymal stem cell differentiation into neurons in vitro (44), suggesting that Notch1 is inhibited during the process of neuronal differentiation. Thus, the Notch1 signaling pathway is considered an important regulator for BMSC differentiation into neurons (4).

The present study examined the association between autophagy and the Notch1 signaling pathway in BMSCs and found that induction of autophagy inhibits Notch1. This affects the ability of BMSCs to differentiate into neurons, which is supported by the evidence that the Notch1 signaling pathway and autophagy are linked (4547). Thus, autophagy may inhibit proliferation and promote differentiation by inhibiting the Notch1 signaling pathway in BMSCs.

The results of the present study demonstrated that induction of autophagy decreased the S-phase population and promoted neuronal differentiation of BMSCs, while inhibition of autophagy reduced apoptosis and neuronal differentiation. These processed were regulated by modulation of the Notch1 signaling pathway. The present study suggested that autophagy has an important role in BMSC proliferation and differentiation. Further study is required to clarify the underlying mechanisms of BMSCs differentiation in vivo.

Acknowledgments

The present study was supported by National Nature Science Foundation of China (grant nos. 81171250 and 31100790).

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February 2016
Volume 13 Issue 2

Print ISSN: 1791-2997
Online ISSN:1791-3004

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APA
Li, B., Duan, P., Li, C., Jing, Y., Han, X., Yan, W., & Xing, Y. (2016). Role of autophagy on bone marrow mesenchymal stem‑cell proliferation and differentiation into neurons. Molecular Medicine Reports, 13, 1413-1419. https://doi.org/10.3892/mmr.2015.4673
MLA
Li, B., Duan, P., Li, C., Jing, Y., Han, X., Yan, W., Xing, Y."Role of autophagy on bone marrow mesenchymal stem‑cell proliferation and differentiation into neurons". Molecular Medicine Reports 13.2 (2016): 1413-1419.
Chicago
Li, B., Duan, P., Li, C., Jing, Y., Han, X., Yan, W., Xing, Y."Role of autophagy on bone marrow mesenchymal stem‑cell proliferation and differentiation into neurons". Molecular Medicine Reports 13, no. 2 (2016): 1413-1419. https://doi.org/10.3892/mmr.2015.4673