Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10320
SHORT COMMUNICATION
Role of autophagy and mTOR signaling in neural differentiation of bone marrow mesenchymal stem cells Yanfei Li, Cuiqin Wang, Guangyu Zhang, Xiaohan Wang, Ranran Duan, Huili Gao, Tao Peng, Junfang Teng and Yanjie Jia* Department of Neurology, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China
Abstract Autophagy is involved in cell differentiation. We present evidence that autophagy is activated during b-mercaptoethanol (bME)-induced neuronal differentiation of bone marrow mesenchymal stem cells (MSCs), in which mammalian target of rapamycin (mTOR) signaling is important. mTOR activity declined after being transported from the nucleus to the cytoplasm. Using 3-methyladenine (3-MA) and rapamycin to regulate the activity of mTOR, it was found that the efficiency of neuronal differentiation was affected. Keywords: autophagy; MSCs; mTOR; neuronal differentiation
Introduction Autophagy is an evolutionarily conserved catabolic program controlled by the autophagy-related family (ATG family) of genes that recycles proteins and organelles. As a consequence of autophagy, cells generate metabolic precursors for macromolecular biosynthesis or ATP generation (He and Klionsky, 2009). Recent studies have shown that autophagy participates in differentiation, a cell remodeling mechanism that promotes morphological and structural changes. Target of rapamycin (TOR) is an evolutionarily conserved protein kinase family and exists widely in all types of biological cells. Mammalian TOR (mTOR) is a member of the phosphatidylinositol kinase-related family of serinethreonine kinases which forms two different complexes: mTOR complex 1 (mTORC1), which is sensitive to rapamycin, and mTOR complex 2 (mTORC2), which is insensitive to rapamycin. mTORC1 contains mTOR, regulatory-associated protein of mTOR (raptor), mammalian ortholog of LST8 (mLST8), and proline-rich Akt substrate of 40 kDa (PRAS40). mTORC2 contains mTOR, mLST8, rapamycin-insensitive companion of mTOR (rictor), and mitogen-activated protein kinase-associated protein 1 (mSIN1). mTORC1 has been shown to regulate cell growth, proliferation and some intracellular activities, such as mRNA transcription, translation and autophagy, whereas
mTORC2 functions in cytoskeleton structure reassembly and cell survival (Loewith et al., 2002). 3-MA is commonly used as a specific inhibitor of autophagic sequestration. Rapamycin, an immunosuppressant drug, can interact with mTORC1 and suppress its kinase activity (Lei et al., 2012). Autophagy is negatively controlled by mTOR, and inhibitors of mTOR act as autophagy inducers. mTOR regulates protein synthesis by phosphorylating two characterized substrates, 70 kDa S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Yonezawa et al., 2004). Microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to autophagosome membranes after processing. Two forms of LC3, called LC3 I and II post-translationally in various cells. The amount of LC3 II is correlated with the extent of autophagosome formation (Kabeya et al., 2000). Bone marrow mesenchymal stem cells (MSCs) have extensive capacity for multilineage differentiation and proliferation. Adult MSCs are able to differentiate into a number of mesenchymal phenotypes, including those that form bone, cartilage, muscle, fat, and other connective tissues (Caplan, 2009). MSCs can differentiate into neurons under particular conditions (Jing et al., 2011). Tau is a type of neuron microtubule-associated protein (MAP), which helps maintain structure of the axon. Microtubule-associated protein-2 (MAP-2) is a kind of protein found specifically in dendritic
Corresponding author: email: [email protected]
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Role of autophagy and mTOR signaling
branching of neurons. Tau and MAP-2 are common biomarkers of neurons. A cell-based therapy for replacing dopaminergic neurons in subjects with Parkinson's disease is the transplantation of differentiated autologous MSCs, which might be a safe and effective approach (Hayashi et al., 2013). We therefore explored the potential roles of autophagy and the mTOR signaling pathway in neuronal differentiation of MSCs. Materials and methods
Cell culture MSCs were obtained from the femurs and tibias of Wistar rats, aged 6–8 weeks. The cells were extracted and cultured in a complete medium consisting of Dulbecco's modified Eagle's medium (DMEM, Invitrogen, USA), 10% fetal bovine serum (FBS, Gibco, USA), and 0.3 mg/mL geneticin. Cells were cultured in a 5% CO2 in air atmosphere at 37 C for 4 days. At confluency, the cells were harvested with 0.25% TrypsinEDTA solution and counted for viable cells using trypan blue, and then plated into 24-well plates at 5 104/well.
Cell grouping MSCs were divided into groups as follows: 3-MA group, rapamycin group and a control group. The concentrations of 3-MA used were 0, 1, 5, and 10 mM and were set as groups 1, 2, 3, 4. The concentrations of rapamycin used were 0, 10, 20 and 30 mM and were set as groups 1, 2, 3, 4. The control group was MSCs with no 3-MA or rapamycin treatment.
Neuronal differentiation of MSCs in vitro To induce neuronal differentiation, subconfluent cultures were treated with pre-induction medium, which consisted of DMEM, 10% FBS and 1 mM b-mercaptoethanol (b-ME) for 24 h. After pre-induction, cells were washed with PBS, and the medium for the 3-MA group was replaced with 3-MA and serum-free medium with 10 mM b-ME for 24 h. The medium for the rapamycin group was replaced with rapamycin and serum-free medium with 10 mM b-ME for 24 h. In the control group, the induction medium was replaced with serum-free medium with 10 mM b-ME for 24 h. The morphology of the MSCs was observed before and after induction using an inverted microscope (Woodbury et al., 2000). To measure the efficiency of neuronal differentiation, 2 neuronal markers, microtubule-associated protein 2 (MAP-2) and tau 24 h were analyzed after induction.
Immunofluorescence
Western blot analysis Cell lysates (100 mL) were collected from each group. Approximately 20 mg of total protein from each lysate was subjected to SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membranes were blocked in 5% non-fat milk for 2 h and incubated at 4 C overnight with one of the following antibodies: LC3B (1:1000, Cell Signaling Technology), MAP-2 (1:1000, Santa Cruz), Tau (1:1000, Santa Cruz), mTOR (1:1000, Cell Signaling Technology), p-mTOR (1:1000, Cell Signaling Technology), p-p70s6k (1:1,000, Cell Signaling Technology), p-4EBP1 (1:1000, Cell Signaling Technology), or b-actin (1:1000, Santa Cruz). The membranes were then incubated with a secondary antibody (1:3000) for 2 h at room temperature and developed using a color reagent. Values were expressed as percentages relative to the loading control, b-actin.
mTOR kinase activity assay We used a K-LISATM (Recombinant) Activity Kit (Millipore, USA) to measure the kinase activity of mTOR. The K-LISATM mTOR (Recombinant) Activity Kit is an ELISA-based activity assay that utilizes a p70S6K-GST fusion protein as a specific mTOR substrate. The mTOR substrate was bound to the wells of a glutathione-coated 96-well plate and then incubated with recombinant mTOR-containing samples. Active mTOR phosphorylates p70S6K at Thr389 in the presence of ATP. The phosphorylated substrate is detected with an antip70S6K-pT389 antibody, followed by detection with an HRP-antibody conjugate and TMB substrate. Sensitivity is increased by the addition of ELISA Stop Solution, and relative activity is determined by absorbance at 450 and 540 nm.
Statistical analysis
After washing with PBS, cells were fixed at 4 C in 4% paraformaldehyde for 20 min, permeabilized with 0.1% 1338
Triton X-100 for 10 min and blocked with 10% bovine serum albumin (BSA) for 1 h. The cells were incubated with the following antibodies: LC3B (LC3 I and II, 1:200, Cell Signaling Technology, USA), LC3 I is cytosolic, whereas LC3 II is membrane bound. Mutational analyses suggest that LC3 I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3 I into LC3 II. MAP-2 (1:200, Santa Cruz, USA), Tau (1:200, Santa Cruz), mTOR (1:200, Cell Signaling Technology) at 4 C overnight. After being washed with PBS, cells were incubated with a secondary antibody (anti-Ig-GCy3 goat anti-rabbit, 1:500, Santa Cruz) at room temperature for 1 h. Cells were observed with an inverted fluorescence microscope.
Each experiment was repeated at least three times. Data are means standard deviation. Statistical differences in
Cell Biol Int 38 (2014) 1337–1343 © 2014 International Federation for Cell Biology
Y. Li et al.
multiple groups were determined by one-way ANOVA. Statistical significance between two groups was determined by Student's t-test using the SPSS software. Differences between groups were considered significant if P < 0.05.
Role of autophagy and mTOR signaling
The abundance of LC3-II or the ratio of LC3-II/LC3-I is a good indicator of autophagosome formation (Levine and Klionsky, 2004). We did not detect LC3-II expression in MSCs before induction, and only a few fluorescent LC3 dots (autophagosomes) were seen by immunofluorescence staining. Both the expression of LC3-II and the ratio of LC3-II/LC3-I were higher after induction (P < 0.05, Figures 1b and 1c). The number of fluorescent LC3 dots also increased after induction (Figure 1a), indicating upregulation of autophagy during differentiation.
mTOR and Western blots to analyze the expression of mTOR, phosphorylated mTOR (p-mTOR) and 2 substrates of mTOR, p-70S6K, and 4E-BP1. Before induction, increasing concentrations of 3-MA (1–10 mM) gradually enhanced mTOR activity. Increasing concentrations of rapamycin (10–30 mM) resulted in a gradual decline in the activity of mTOR. After induction, similar results were obtained. The activity of mTOR was also lower after than before induction with no rapamycin or 3-MA treatment. Overall, mTOR activity declined after induction compared to pre-induction (P < 0.05, Figure 2h). Before induction, the expression of p-mTOR, p-p70S6K, and p-4E-BP1 was increased with increasing concentrations of 3-MA, but rapamycin had the opposite effect. Total expression of mTOR was unchanged. Using 3-MA or rapamycin, similar expression patterns of associated phosphorylation proteins after induction were observed. It can be concluded that the expression of p-mTOR, p-p70S6K, and p-4E-BP1 after induction, declined (P < 0.05, Figures 2a–2g, 2i and 2j).
Downregulation of mTOR signaling during the neuronal differentiation of MSCs
An appropriate level of mTOR activity is important for neuronal differentiation of MSCs
To explore the possible mechanism by which autophagy was activated during neuronal differentiation of MSCs, we examined mTOR signaling activity 24 h after bmercaptoethanol (b-ME) induction. The K-LISATM mTOR (Recombinant) Activity Kit was used to detect
Considering that mTOR is critical in neuronal differentiation, we tested whether changes in mTOR activity affected differentiation. To examine the efficiency of differentiation, 2 neuronal markers, microtubule-associated protein 2 (MAP-2) and tau, were analyzed 24 h after induction.
Results
Autophagy was activated during the neuronal differentiation of MSCs
Figure 1 Autophagy activity during the neuronal differentiation of MSCs. Autophagy activity increased during the process of neuronal differentiation of MSCs: (a) LC3-positive dots (in red) in MSCs (scale bar: 20 mm). The number of fluorescent LC3 dots increased after 24 h induction. (b) Autophagy-dependent expression of LC3-II in MSCs. (c) Protein levels of LC3 were analyzed by Western blotting. The expression of LC3-II and the ratio of LC3-II/LC3-I was higher after induction of the neuronal differentiation of MSCs (n ¼ 3, P < 0.05).
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Figure 2 Downregulation of mTOR signaling. (a) The expression levels of mTOR (b), p-mTOR (b), p-p70S6K (b), and p-4E-BP1 (b) were analyzed by Western blotting techniques prior to induction with b-ME. The expression levels of mTOR (a), p-mTOR (a), pp70S6K (a), and p-4E-BP1 (a) were analyzed by Western blotting techniques 24 h after induction with b-ME. The expression of b-actin was used as a loading control. (b–g and i and j): Quantification of the expression levels of mTOR, p-mTOR, p-p70S6K, and p-4E-BP1 (mean SD). Before induction, the expression levels of p-mTOR, p-p70S6K, and p-4E-BP1 were elevated with increasing concentrations of 3-MA and were decreased with increasing concentrations of rapamycin. The expression of p-mTOR, p-p70S6K, and p-4E-BP1 declined after induction compared to before induction (n ¼ 3, P < 0.05). (h): The activity of mTOR was analyzed by the K-LISA assay before and after induction. mTOR activity declined after induction compared to preinduction (n ¼ 3, P < 0.05).
One day after induction, rising concentrations of rapamycin (10–20 mM) gradually enhanced the fluorescence signal of neurite elongation (Figures 3b and 3c, 3i and 3j). When the concentration reached 30 mM, the fluorescence signal was weakened (Figures 3d and 3k). 3-MA (1–10 mM) caused a gradual weakening in fluorescence signal of neurite elongation (Figures 3e–3g, 3l–3n). The expression of MAP-2 and tau in the rapamycin 20 mM group was the highest compared to the other groups (P < 0.05, Figures 3o–3q). 1340
Cytoplasmic nuclear shuttling of mTOR during the neuronal differentiation of MSCs Before induction, the fluorescence signal was mainly distributed in the nucleus; it was enhanced in the 3-MA group (Figures 4d–4f), but weakened in the rapamycin group (Figures 4a–4c) compared to the control group. After induction, the fluorescence signal in the nucleus weakened in the rapamycin group compared (Figures 4g–4i). In the
Cell Biol Int 38 (2014) 1337–1343 © 2014 International Federation for Cell Biology
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Role of autophagy and mTOR signaling
Figure 3 Expression of Tau and MAP-2. (a–g): The expression levels of MAP-2 by immunofluorescence staining (scale bar: 50 mm). (h–n): The expression levels of Tau by immunofluorescence staining (scale bar: 50 mm). (o): The expression levels of MAP-2 and Tau were analyzed by Western blotting analysis 24 h after induction with b-ME. (p and q): The quantification of the expression levels of MAP-2 and Tau (mean SD, n ¼ 6). rapamycin: 1 (0 mM), 2 (10 mM), 3 (20 mM), 4 (30 mM). 3-MA: 1 (0 mM), 2 (1 mM), 3 (5 mM), 4 (10 mM).
3-MA groups, there was an obvious fluorescence signal accumulating in the nucleus (Figures 4j–4l). Discussion Zhao et al. (2010) found that autophagy activity was markedly increased when glioma stem/progenitor cells (GSPCs) were induced to differentiate using fetal calf serum (FCS), and that rapamycin, the activator of autophagy, could promote their differentiation. The mTOR signaling pathway was involved the regulation of autophagy, and mTOR could turn into its activated form, phospho-mTOR, which can respond directly to mTOR activity (Fielhaber et al., 2012). Our study demonstrated that the expression of p-mTOR, pp70S6K, and p-4E-BP1 declined after induction compared, which indicates that mTOR activity decreased during the neuronal differentiation. mTOR activity is modified in various pathological states of the nervous system, including brain tumors, tuberous sclerosis and neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases (Swiech et al., 2008). mTOR is important in the regulation of neural differentiation and synaptic plasticity (Magri et al., 2011). In some treatments for neurodegenerative disorders, decrease in neuronal mTOR activity can alleviate disease progression. mTOR activity that is either too low or too high impairs cell differentiation. High concentration rapamycin in neuronal differentiation can reduce nerve swelling, cell size and neural markers of immune activity (Zeng and Zhou, 2008). We
found that rising concentrations of rapamycin (10–20 mM) gradually increased the expression of MAP-2 and tau after induction; when it reached 30 mM, MAP-2 and tau expression declined, which indicates an appropriate level of mTOR activity be needed in neuronal differentiation. In human embryonic kidney (HEK) 293 cells, mTOR is a cytoplasmic-nuclear shuttling protein, which may be relevant to the mitogen-stimulated rapamycin-sensitive mTOR signaling pathway and translation initiation (Kim and Chen, 2000). mTOR shuttled from its normal predominantly mitochondrial location to the cell nucleus under certain conditions (Tirado et al., 2003; Bachmann et al., 2006). Altering mTOR nuclear shuttling with exogenous nuclear import and export signals has shown that they are consistent with a direct link between the nuclear shuttling of mTOR and the mitogenic stimulation of p70S6K activation and 4EBP1 phosphorylation. Before induction, the fluorescence signal is mainly distributed in the nucleus and this signal is enhanced in the 3-MA group, but weakened in the rapamycin group. After induction, the fluorescence signal in the nucleus weakened in the rapamycin group and there was an obvious fluorescence signal accumulating in the nucleus in the 3-MA groups, which indicates nuclear shuttling of mTOR might occur in neuronal differentiation. During the neuronal differentiation of MSCs, autophagy activity increased. The expression of mTOR signaling was downregulated during differentiation. Neuronal differentiation required proper mTOR activity, elevated or downregulated mTOR activity could perturb axon growth and
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Figure 4 Subcellular distribution of mTOR. The expression levels of mTOR (red) and DAPI (blue) by immunofluorescence staining (scale bar: 250 mm). (a–f): Before induction. (g–l): Twenty-four hours after induction with b-ME. (a–c and g–i): Rapamycin 10, 20, 30 mM; (d–f and j–l): 3-MA 1, 5, 10 mM.
navigation, dendritic arborization, and synaptogenesis. In conclusion, autophagy is activated and might be important in neuronal differentiation of MSCs. Neuronal differentiation appears to require normal mTOR activity. Acknowledgment and funding This work was supported by the National Natural Science Foundation of China (Grant Numbers 81071114). 1342
References Bachmann RA, Kim JH, Wu AL, Park IH, Chen J (2006) A nuclear transport signal in mammalian target of rapamycin is critical for its cytoplasmic signaling to S6 kinase 1. J Biol Chem 281: 7357–63. Caplan AI (2009) Why are MSCs therapeutic? New data: new insight. J Pahol 217: 318–24. Fielhaber JA, Tan J, Joung KB, Attias O, Huegel S, Bader M, Roux PP, Kristof AS (2012) Regulation of karyopherin a1 and
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nuclear import by mammalian target of rapamycin. J Biol Chem 287: 14325–35. Hayashi T, Wakao S, Kitada M, Ose T, Watabe H, Kuroda Y, Mitsunaga K, Matsuse D, Shigemoto T, Ito A, Ikeda H, Fukuyama H, Onoe H, Tabata Y, Dezawa M (2013) Autologous mesenchymal stem cell-derived dopaminergic neurons function in parkinsonian macaques. J Clin Invest 123: 272–84. He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43: 67–93. Jing L, Jia Y, Lu J, Han R, Li J, Wang S, Peng T, Jia Y (2011) MicroRNA-9 promotes differentiation of mouse bone mesenchymal stem cells into neurons by Notch signaling. Neuroreport 22: 206–11. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19: 5720–8. Kim JE, Chen J (2000) Cytoplasmic-nuclear shuttling of FKBP12rapamycin associated protein is involved in rapamycinsensitive signaling and translation initiation. Proc Natl Acad Sci USA 97: 14340–5. Lei FR, Li XQ, Liu H, Zhu RD, Meng QY, Rong JJ (2012) Rapamycin and 3-methyladenine regulate apoptosis and autophagy in bone-derived endothelial progenitor cells. Chin Med J 125: 4076–82. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6: 463–77. Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, Bonenfant D, Oppliger W, Jenoe P, Hall MN (2002) Two TOR
Role of autophagy and mTOR signaling
complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 10: 457–68. Magri L, Cambiaghi M, Cominelli M, Alfaro-Cervello C, Cursi M, Pala M, Bulfone A, Garcìa-Verdugo JM, Leocani L, Minicucci F, Poliani PL, Galli R (2011) Sustained activation of mTOR pathway in embryonic neural stem cells leads to development of tuberous scleresis complex-associated lesions. Cell Stem Cell 9: 447–62. Swiech L, Perycz M, Malik A, Jaworski J (2008) Role of mTOR in physiology and pathology of the nervous system. Biochim Biophys Acta 1784: 116–32. Tirado OM, Mateo-Lozano S, Sanders S, Dettin LE, Notario V (2003) The PCPH oncoprotein antagonizes the proapoptotic role of the mammalian target of rapamycin in the response of normal fibroblast to ionizing radiation. Cancer Res 63: 6290–8. Woodbury D, Schwarz EJ, Prockop DJ, Black IB (2000) Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61: 364–70. Yonezawa K, Yoshino KI, Tokunaga C, Hara K (2004) Kinase activities associated with mTOR. Curr Top Microbiol Immunol 279: 271–82. Zeng M, Zhou JN (2008) Roles of autophagy and mTOR signaling in neural differentiation of mouse neuroblastoma cells. Cell Signal 20: 659–65. Zhao Y, Huang Q, Yang J, Lou M, Wang A, Dong J, Qin Z, Zhang T (2010) Autophagy impairment inhibits differentiation of glioma stem/progenitor cells. Brain Res 1313: 250–8. Received 20 January 2014; accepted 25 April 2014. Final version published online 18 August 2014.
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SHORT COMMUNICATION
Role of autophagy and mTOR signaling in neural differentiation of bone marrow mesenchymal stem cells Yanfei Li, Cuiqin Wang, Guangyu Zhang, Xiaohan Wang, Ranran Duan, Huili Gao, Tao Peng, Junfang Teng and Yanjie Jia* Department of Neurology, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China
Abstract Autophagy is involved in cell differentiation. We present evidence that autophagy is activated during b-mercaptoethanol (bME)-induced neuronal differentiation of bone marrow mesenchymal stem cells (MSCs), in which mammalian target of rapamycin (mTOR) signaling is important. mTOR activity declined after being transported from the nucleus to the cytoplasm. Using 3-methyladenine (3-MA) and rapamycin to regulate the activity of mTOR, it was found that the efficiency of neuronal differentiation was affected. Keywords: autophagy; MSCs; mTOR; neuronal differentiation
Introduction Autophagy is an evolutionarily conserved catabolic program controlled by the autophagy-related family (ATG family) of genes that recycles proteins and organelles. As a consequence of autophagy, cells generate metabolic precursors for macromolecular biosynthesis or ATP generation (He and Klionsky, 2009). Recent studies have shown that autophagy participates in differentiation, a cell remodeling mechanism that promotes morphological and structural changes. Target of rapamycin (TOR) is an evolutionarily conserved protein kinase family and exists widely in all types of biological cells. Mammalian TOR (mTOR) is a member of the phosphatidylinositol kinase-related family of serinethreonine kinases which forms two different complexes: mTOR complex 1 (mTORC1), which is sensitive to rapamycin, and mTOR complex 2 (mTORC2), which is insensitive to rapamycin. mTORC1 contains mTOR, regulatory-associated protein of mTOR (raptor), mammalian ortholog of LST8 (mLST8), and proline-rich Akt substrate of 40 kDa (PRAS40). mTORC2 contains mTOR, mLST8, rapamycin-insensitive companion of mTOR (rictor), and mitogen-activated protein kinase-associated protein 1 (mSIN1). mTORC1 has been shown to regulate cell growth, proliferation and some intracellular activities, such as mRNA transcription, translation and autophagy, whereas
mTORC2 functions in cytoskeleton structure reassembly and cell survival (Loewith et al., 2002). 3-MA is commonly used as a specific inhibitor of autophagic sequestration. Rapamycin, an immunosuppressant drug, can interact with mTORC1 and suppress its kinase activity (Lei et al., 2012). Autophagy is negatively controlled by mTOR, and inhibitors of mTOR act as autophagy inducers. mTOR regulates protein synthesis by phosphorylating two characterized substrates, 70 kDa S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Yonezawa et al., 2004). Microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to autophagosome membranes after processing. Two forms of LC3, called LC3 I and II post-translationally in various cells. The amount of LC3 II is correlated with the extent of autophagosome formation (Kabeya et al., 2000). Bone marrow mesenchymal stem cells (MSCs) have extensive capacity for multilineage differentiation and proliferation. Adult MSCs are able to differentiate into a number of mesenchymal phenotypes, including those that form bone, cartilage, muscle, fat, and other connective tissues (Caplan, 2009). MSCs can differentiate into neurons under particular conditions (Jing et al., 2011). Tau is a type of neuron microtubule-associated protein (MAP), which helps maintain structure of the axon. Microtubule-associated protein-2 (MAP-2) is a kind of protein found specifically in dendritic
Corresponding author: email: [email protected]
Cell Biol Int 38 (2014) 1337–1343 © 2014 International Federation for Cell Biology
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Y. Li et al.
Role of autophagy and mTOR signaling
branching of neurons. Tau and MAP-2 are common biomarkers of neurons. A cell-based therapy for replacing dopaminergic neurons in subjects with Parkinson's disease is the transplantation of differentiated autologous MSCs, which might be a safe and effective approach (Hayashi et al., 2013). We therefore explored the potential roles of autophagy and the mTOR signaling pathway in neuronal differentiation of MSCs. Materials and methods
Cell culture MSCs were obtained from the femurs and tibias of Wistar rats, aged 6–8 weeks. The cells were extracted and cultured in a complete medium consisting of Dulbecco's modified Eagle's medium (DMEM, Invitrogen, USA), 10% fetal bovine serum (FBS, Gibco, USA), and 0.3 mg/mL geneticin. Cells were cultured in a 5% CO2 in air atmosphere at 37 C for 4 days. At confluency, the cells were harvested with 0.25% TrypsinEDTA solution and counted for viable cells using trypan blue, and then plated into 24-well plates at 5 104/well.
Cell grouping MSCs were divided into groups as follows: 3-MA group, rapamycin group and a control group. The concentrations of 3-MA used were 0, 1, 5, and 10 mM and were set as groups 1, 2, 3, 4. The concentrations of rapamycin used were 0, 10, 20 and 30 mM and were set as groups 1, 2, 3, 4. The control group was MSCs with no 3-MA or rapamycin treatment.
Neuronal differentiation of MSCs in vitro To induce neuronal differentiation, subconfluent cultures were treated with pre-induction medium, which consisted of DMEM, 10% FBS and 1 mM b-mercaptoethanol (b-ME) for 24 h. After pre-induction, cells were washed with PBS, and the medium for the 3-MA group was replaced with 3-MA and serum-free medium with 10 mM b-ME for 24 h. The medium for the rapamycin group was replaced with rapamycin and serum-free medium with 10 mM b-ME for 24 h. In the control group, the induction medium was replaced with serum-free medium with 10 mM b-ME for 24 h. The morphology of the MSCs was observed before and after induction using an inverted microscope (Woodbury et al., 2000). To measure the efficiency of neuronal differentiation, 2 neuronal markers, microtubule-associated protein 2 (MAP-2) and tau 24 h were analyzed after induction.
Immunofluorescence
Western blot analysis Cell lysates (100 mL) were collected from each group. Approximately 20 mg of total protein from each lysate was subjected to SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membranes were blocked in 5% non-fat milk for 2 h and incubated at 4 C overnight with one of the following antibodies: LC3B (1:1000, Cell Signaling Technology), MAP-2 (1:1000, Santa Cruz), Tau (1:1000, Santa Cruz), mTOR (1:1000, Cell Signaling Technology), p-mTOR (1:1000, Cell Signaling Technology), p-p70s6k (1:1,000, Cell Signaling Technology), p-4EBP1 (1:1000, Cell Signaling Technology), or b-actin (1:1000, Santa Cruz). The membranes were then incubated with a secondary antibody (1:3000) for 2 h at room temperature and developed using a color reagent. Values were expressed as percentages relative to the loading control, b-actin.
mTOR kinase activity assay We used a K-LISATM (Recombinant) Activity Kit (Millipore, USA) to measure the kinase activity of mTOR. The K-LISATM mTOR (Recombinant) Activity Kit is an ELISA-based activity assay that utilizes a p70S6K-GST fusion protein as a specific mTOR substrate. The mTOR substrate was bound to the wells of a glutathione-coated 96-well plate and then incubated with recombinant mTOR-containing samples. Active mTOR phosphorylates p70S6K at Thr389 in the presence of ATP. The phosphorylated substrate is detected with an antip70S6K-pT389 antibody, followed by detection with an HRP-antibody conjugate and TMB substrate. Sensitivity is increased by the addition of ELISA Stop Solution, and relative activity is determined by absorbance at 450 and 540 nm.
Statistical analysis
After washing with PBS, cells were fixed at 4 C in 4% paraformaldehyde for 20 min, permeabilized with 0.1% 1338
Triton X-100 for 10 min and blocked with 10% bovine serum albumin (BSA) for 1 h. The cells were incubated with the following antibodies: LC3B (LC3 I and II, 1:200, Cell Signaling Technology, USA), LC3 I is cytosolic, whereas LC3 II is membrane bound. Mutational analyses suggest that LC3 I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3 I into LC3 II. MAP-2 (1:200, Santa Cruz, USA), Tau (1:200, Santa Cruz), mTOR (1:200, Cell Signaling Technology) at 4 C overnight. After being washed with PBS, cells were incubated with a secondary antibody (anti-Ig-GCy3 goat anti-rabbit, 1:500, Santa Cruz) at room temperature for 1 h. Cells were observed with an inverted fluorescence microscope.
Each experiment was repeated at least three times. Data are means standard deviation. Statistical differences in
Cell Biol Int 38 (2014) 1337–1343 © 2014 International Federation for Cell Biology
Y. Li et al.
multiple groups were determined by one-way ANOVA. Statistical significance between two groups was determined by Student's t-test using the SPSS software. Differences between groups were considered significant if P < 0.05.
Role of autophagy and mTOR signaling
The abundance of LC3-II or the ratio of LC3-II/LC3-I is a good indicator of autophagosome formation (Levine and Klionsky, 2004). We did not detect LC3-II expression in MSCs before induction, and only a few fluorescent LC3 dots (autophagosomes) were seen by immunofluorescence staining. Both the expression of LC3-II and the ratio of LC3-II/LC3-I were higher after induction (P < 0.05, Figures 1b and 1c). The number of fluorescent LC3 dots also increased after induction (Figure 1a), indicating upregulation of autophagy during differentiation.
mTOR and Western blots to analyze the expression of mTOR, phosphorylated mTOR (p-mTOR) and 2 substrates of mTOR, p-70S6K, and 4E-BP1. Before induction, increasing concentrations of 3-MA (1–10 mM) gradually enhanced mTOR activity. Increasing concentrations of rapamycin (10–30 mM) resulted in a gradual decline in the activity of mTOR. After induction, similar results were obtained. The activity of mTOR was also lower after than before induction with no rapamycin or 3-MA treatment. Overall, mTOR activity declined after induction compared to pre-induction (P < 0.05, Figure 2h). Before induction, the expression of p-mTOR, p-p70S6K, and p-4E-BP1 was increased with increasing concentrations of 3-MA, but rapamycin had the opposite effect. Total expression of mTOR was unchanged. Using 3-MA or rapamycin, similar expression patterns of associated phosphorylation proteins after induction were observed. It can be concluded that the expression of p-mTOR, p-p70S6K, and p-4E-BP1 after induction, declined (P < 0.05, Figures 2a–2g, 2i and 2j).
Downregulation of mTOR signaling during the neuronal differentiation of MSCs
An appropriate level of mTOR activity is important for neuronal differentiation of MSCs
To explore the possible mechanism by which autophagy was activated during neuronal differentiation of MSCs, we examined mTOR signaling activity 24 h after bmercaptoethanol (b-ME) induction. The K-LISATM mTOR (Recombinant) Activity Kit was used to detect
Considering that mTOR is critical in neuronal differentiation, we tested whether changes in mTOR activity affected differentiation. To examine the efficiency of differentiation, 2 neuronal markers, microtubule-associated protein 2 (MAP-2) and tau, were analyzed 24 h after induction.
Results
Autophagy was activated during the neuronal differentiation of MSCs
Figure 1 Autophagy activity during the neuronal differentiation of MSCs. Autophagy activity increased during the process of neuronal differentiation of MSCs: (a) LC3-positive dots (in red) in MSCs (scale bar: 20 mm). The number of fluorescent LC3 dots increased after 24 h induction. (b) Autophagy-dependent expression of LC3-II in MSCs. (c) Protein levels of LC3 were analyzed by Western blotting. The expression of LC3-II and the ratio of LC3-II/LC3-I was higher after induction of the neuronal differentiation of MSCs (n ¼ 3, P < 0.05).
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Role of autophagy and mTOR signaling
Figure 2 Downregulation of mTOR signaling. (a) The expression levels of mTOR (b), p-mTOR (b), p-p70S6K (b), and p-4E-BP1 (b) were analyzed by Western blotting techniques prior to induction with b-ME. The expression levels of mTOR (a), p-mTOR (a), pp70S6K (a), and p-4E-BP1 (a) were analyzed by Western blotting techniques 24 h after induction with b-ME. The expression of b-actin was used as a loading control. (b–g and i and j): Quantification of the expression levels of mTOR, p-mTOR, p-p70S6K, and p-4E-BP1 (mean SD). Before induction, the expression levels of p-mTOR, p-p70S6K, and p-4E-BP1 were elevated with increasing concentrations of 3-MA and were decreased with increasing concentrations of rapamycin. The expression of p-mTOR, p-p70S6K, and p-4E-BP1 declined after induction compared to before induction (n ¼ 3, P < 0.05). (h): The activity of mTOR was analyzed by the K-LISA assay before and after induction. mTOR activity declined after induction compared to preinduction (n ¼ 3, P < 0.05).
One day after induction, rising concentrations of rapamycin (10–20 mM) gradually enhanced the fluorescence signal of neurite elongation (Figures 3b and 3c, 3i and 3j). When the concentration reached 30 mM, the fluorescence signal was weakened (Figures 3d and 3k). 3-MA (1–10 mM) caused a gradual weakening in fluorescence signal of neurite elongation (Figures 3e–3g, 3l–3n). The expression of MAP-2 and tau in the rapamycin 20 mM group was the highest compared to the other groups (P < 0.05, Figures 3o–3q). 1340
Cytoplasmic nuclear shuttling of mTOR during the neuronal differentiation of MSCs Before induction, the fluorescence signal was mainly distributed in the nucleus; it was enhanced in the 3-MA group (Figures 4d–4f), but weakened in the rapamycin group (Figures 4a–4c) compared to the control group. After induction, the fluorescence signal in the nucleus weakened in the rapamycin group compared (Figures 4g–4i). In the
Cell Biol Int 38 (2014) 1337–1343 © 2014 International Federation for Cell Biology
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Role of autophagy and mTOR signaling
Figure 3 Expression of Tau and MAP-2. (a–g): The expression levels of MAP-2 by immunofluorescence staining (scale bar: 50 mm). (h–n): The expression levels of Tau by immunofluorescence staining (scale bar: 50 mm). (o): The expression levels of MAP-2 and Tau were analyzed by Western blotting analysis 24 h after induction with b-ME. (p and q): The quantification of the expression levels of MAP-2 and Tau (mean SD, n ¼ 6). rapamycin: 1 (0 mM), 2 (10 mM), 3 (20 mM), 4 (30 mM). 3-MA: 1 (0 mM), 2 (1 mM), 3 (5 mM), 4 (10 mM).
3-MA groups, there was an obvious fluorescence signal accumulating in the nucleus (Figures 4j–4l). Discussion Zhao et al. (2010) found that autophagy activity was markedly increased when glioma stem/progenitor cells (GSPCs) were induced to differentiate using fetal calf serum (FCS), and that rapamycin, the activator of autophagy, could promote their differentiation. The mTOR signaling pathway was involved the regulation of autophagy, and mTOR could turn into its activated form, phospho-mTOR, which can respond directly to mTOR activity (Fielhaber et al., 2012). Our study demonstrated that the expression of p-mTOR, pp70S6K, and p-4E-BP1 declined after induction compared, which indicates that mTOR activity decreased during the neuronal differentiation. mTOR activity is modified in various pathological states of the nervous system, including brain tumors, tuberous sclerosis and neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases (Swiech et al., 2008). mTOR is important in the regulation of neural differentiation and synaptic plasticity (Magri et al., 2011). In some treatments for neurodegenerative disorders, decrease in neuronal mTOR activity can alleviate disease progression. mTOR activity that is either too low or too high impairs cell differentiation. High concentration rapamycin in neuronal differentiation can reduce nerve swelling, cell size and neural markers of immune activity (Zeng and Zhou, 2008). We
found that rising concentrations of rapamycin (10–20 mM) gradually increased the expression of MAP-2 and tau after induction; when it reached 30 mM, MAP-2 and tau expression declined, which indicates an appropriate level of mTOR activity be needed in neuronal differentiation. In human embryonic kidney (HEK) 293 cells, mTOR is a cytoplasmic-nuclear shuttling protein, which may be relevant to the mitogen-stimulated rapamycin-sensitive mTOR signaling pathway and translation initiation (Kim and Chen, 2000). mTOR shuttled from its normal predominantly mitochondrial location to the cell nucleus under certain conditions (Tirado et al., 2003; Bachmann et al., 2006). Altering mTOR nuclear shuttling with exogenous nuclear import and export signals has shown that they are consistent with a direct link between the nuclear shuttling of mTOR and the mitogenic stimulation of p70S6K activation and 4EBP1 phosphorylation. Before induction, the fluorescence signal is mainly distributed in the nucleus and this signal is enhanced in the 3-MA group, but weakened in the rapamycin group. After induction, the fluorescence signal in the nucleus weakened in the rapamycin group and there was an obvious fluorescence signal accumulating in the nucleus in the 3-MA groups, which indicates nuclear shuttling of mTOR might occur in neuronal differentiation. During the neuronal differentiation of MSCs, autophagy activity increased. The expression of mTOR signaling was downregulated during differentiation. Neuronal differentiation required proper mTOR activity, elevated or downregulated mTOR activity could perturb axon growth and
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Role of autophagy and mTOR signaling
Figure 4 Subcellular distribution of mTOR. The expression levels of mTOR (red) and DAPI (blue) by immunofluorescence staining (scale bar: 250 mm). (a–f): Before induction. (g–l): Twenty-four hours after induction with b-ME. (a–c and g–i): Rapamycin 10, 20, 30 mM; (d–f and j–l): 3-MA 1, 5, 10 mM.
navigation, dendritic arborization, and synaptogenesis. In conclusion, autophagy is activated and might be important in neuronal differentiation of MSCs. Neuronal differentiation appears to require normal mTOR activity. Acknowledgment and funding This work was supported by the National Natural Science Foundation of China (Grant Numbers 81071114). 1342
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Role of autophagy and mTOR signaling
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