Downregulation of APE1/Ref-1 Is Involved in the Senescence of Mesenchymal Stem Cells

Authors

  • Jun-Young Heo,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
    2. Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Kaipeng Jing,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Kyoung-Sub Song,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Kang-Sik Seo,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Ji-Hoon Park,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Jong-Seok Kim,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Yeon-Joo Jung,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Gang-Min Hur,

    1. Department of Pharmacology, College of Medicine, Chungnam National University, Teajeon, South Korea
    2. Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Teajeon, South Korea
    3. Research Institutes of Biomedical Sciences, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Deog-Yeon Jo,

    1. Division of Hematology/Oncology Department of Internal Medicine, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Gi-Ryang Kweon,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
    2. Research Institutes of Biomedical Sciences, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Wan-Hee Yoon,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Kyu Lim,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Byung-Doo Hwang,

    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
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  • Byeong Hwa Jeon,

    Corresponding author
    1. Department of Physiology, College of Medicine, Chungnam National University, Teajeon, South Korea
    2. Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Teajeon, South Korea
    3. Research Institutes of Biomedical Sciences, College of Medicine, Chungnam National University, Teajeon, South Korea
    • Byeong Hwa Jeon, Department of Physiology, College of Medicine, Chungnam National University, 6, Munhwa-Dong, Joong-Gu, Teajeon, 301-747, South Korea

      Jong-Il Park, Department of Biochemistry, College of Medicine, Chungnam National University, 6, Munhwa-Dong, Joong-Gu, Teajeon, 301-747, South Korea

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    • Telephone: 82-42-580-8214

  • Jong-Il Park

    Corresponding author
    1. Department of Biochemistry, College of Medicine, Chungnam National University, Teajeon, South Korea
    2. Research Institutes of Biomedical Sciences, College of Medicine, Chungnam National University, Teajeon, South Korea
    • Byeong Hwa Jeon, Department of Physiology, College of Medicine, Chungnam National University, 6, Munhwa-Dong, Joong-Gu, Teajeon, 301-747, South Korea

      Jong-Il Park, Department of Biochemistry, College of Medicine, Chungnam National University, 6, Munhwa-Dong, Joong-Gu, Teajeon, 301-747, South Korea

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    • Telephone: 82-42-580-8225; Fax: 82-42-580-8121


  • Author contributions: J.-Y.H., K.J., K.-S. Song, K.-S. Seo, J.-H.P., J.-S.K.: collection and assembly of data; Y.-J.J.: Provision of study material; G.-M.H., D.-Y.J., G.-R.K., W.-H.Y., K.L., B.-D.H.: Data analysis and interpretation; B.H.J. and J.-I.P.: conception and design; J.-Y.H.: manuscript writing.

  • First published online in STEM CELLS Express March 5, 2009

Abstract

The senescence of human mesenchymal stem cells (hMSCs) causes disruption of tissue and organ maintenance, and is thus an obstacle to stem cell-based therapies for disease. Although some researchers have studied changes in the characteristics of hMSCs (decreases in differentiation ability and self-renewal), comparing young and old ages, the mechanisms of stem cell senescence have not yet been defined. In this study, we developed a growth curve for human bone marrow derived MSCs (hBMSCs) which changes into a hyperbolic state after passage number 7. Senescence associated β-galactosidase (SA β-gal) staining of hBMSCs showed 10% in passage 9 and 45% in passage 11. We detected an increase in endogenous superoxide levels during senescence that correlated with senescence markers (SA β-gal, hyperbolic growth curve). Interestingly, even though endogenous superoxide increased in a replicative senescence model, the expression of APE1/Ref-1, which is sensitive to intracellular redox state, decreased. These effects were confirmed in a stress-induced senescence model by exogenous treatment with H2O2. This change is related to the p53 activity that negatively regulates APE1/Ref-1. p21 expression levels, which represent p53 activity, were transiently increased in passage 9, meaning that they correlated with the expression of APE1/Ref-1. Overexpression of APE1/Ref-1 suppressed superoxide production and decreased SA β-gal in hBMSCs. In conclusion, intracellular superoxide accumulation appears to be the main cause of the senescence of hBMSCs, and overexpression of APE1/Ref-1 can rescue cells from the senescence phenotype. Maintaining characteristics of hBMSCs by regulating intracellular reactive oxygen species production can contribute to tissue regeneration and to improved cell therapy. STEM CELLS 2009;27:1455–1462

INTRODUCTION

Mesenchymal stem cells (MSCs) maintain the ability to replicate due to their long-term self-renewal potential and their ability to differentiate into multiple lineages are a renewable source of replacement cells and tissues to treat myriad diseases and disabilities, including heart disease, osteoarthritis, rheumatoid arthritis, and tendon rupture [1]. However, MSCs undergo an alteration in their physiological properties during in vitro culture and the mechanisms underlying this transition remain elusive.

Senescence has been reported to occur during in vitro expansion of stem cell populations, but the underlying mechanism is not clear. The signs of senescence include a diminishing ability to undergo cell division, an increase in cell size, increased actin stress fiber, and increased β-galactosidase activity in vitro [2]. Isolated MSCs from older donors have a similar phenotype to in vitro senescent cells. The ability to proliferate is decreased, cell size is enlarged, there are increases in the levels of biomarkers of aging and increases in levels of reactive oxygen species (ROSs) in older age groups compared with younger age groups [3].

ROSs have roles as mediators of growth signaling for induction of cell proliferation, in responses to stress, and in energy metabolism [4, 5]. In the case of MSCs, ROSs are also involved in signaling, proliferation, and differentiation [6, 7]. However, if ROS accumulate in the cell due to extended oxidative stress or irreparable DNA damage, they can lead to apoptotic cell death or to senescence through the p53 pathway [8, 9]. The p53 tumor suppressor gene is involved in cell cycle regulation, apoptosis, senescence, and differentiation in several biological systems. Some researchers have suggested that p53 has a role in aging because p53 induces expression of p16 in senescent cells. But others consider p53 to be a sensor for ROS thus having a role in antiageing [10–12]. Because of inducing the expression of antioxidant genes (sestrin, gpx1, adh1), a basal level of p53 can be a primary barrier to DNA damage [13]. Numerous articles have reported that p21 has a role in maintaining long-term self-renewal of stem cells, and prevent stem cell exhaustion and cell cycle arrest [11, 14].

To prevent ROS accumulation, cells have several antioxidant systems including enzymes such as superoxide dismutase, catalase, and glutathione peroxidase which transform ROS to H2O2. Besides antioxidant enzymes, redox-sensitive molecules (Trx, APE1/Ref-1) protect the cell from oxidative stress [15]. The apurinic/apyrimidinic endonuclease1/redox factor-1 (APE1/Ref-1) decreases intracellular ROS by inhibiting rac1 regulated NAD(P)H oxidase [16]. Activation of APE1/Ref-1 increases the binding of oxidative stress regulating transcription factors (Hif-1, p53, Ap-1). In aged rats, APE1/Ref-1 is decreased and responses to stress in the brain are defective [17, 18]. Hence, we can postulate that APE1/Ref-1 regulates the proliferation of MSCs by preventing ROS accumulation.

There are many studies on the relationship between p53 and APE1/Ref-1 [19, 20]. Oxidized APE1/Ref-1 facilitates p53 DNA binding. In contrast, p53 is a negative regulator of APE1/Ref-1 interfering sp1 binding to the APE1/Ref-1 promoter.

Herein we present the relationship between the oxidative stress of human bone marrow derived MSCs (hBMSCs) aging and APE1/Ref-1. In addition, we examined p53 activity during senescence of hBMSCs. We predicted APE1/Ref-1 would have an antisenescence effect via effects on fundamental cellular antioxidant system.

MATERIALS AND METHODS

Isolation and Culture of hBMSCs

Bone marrow (BM) samples were obtained with informed consent (according to the ethical standard of the local ethical committee and with the Declarations of Helsinki in 1995) from three healthy donors undergoing BM harvest. hBMSCs were isolated as previously described [21]. Briefly, mononuclear cell (MNC) fraction was isolated by Percoll density gradient centrifugation. MNC was seeded at an Iscove's modified Dulbecco's medium (IMDM) with 20% heat inactivated fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml). After 7–10 days, trypsinized and replated in a new culture flask. The cells (passage number 1) were incubated at 37°C under air with 5% CO2. The medium was replaced every 3–4 days. At 80% confluence, cells were removed with 0.025% Trypsin-EDTA and placed in fresh culture plates. We confirmed the characterization of our cells using a mesenchymal stem cell kit (Chemicon, Temecula, CA, http://www.chemicon.com). The potential for differentiation into mesodermal lineages was confirmed by a previously described method [22]. These results strongly suggest that MSCs used in this experiment have natural character of human mesenchymal stem cell that is consistent with other reported hBMSCs by adding to the supporting information Figure 1.

Measurement of the Time Required for Population Doublings

Cells (4 × 104 cells per six-well culture plate) were cultured at 37°C under 5% CO2 in a humidified incubator. When subcultures reached at 80–90% confluence, cells underwent trypsinization. Population doublings (PDs) were calculated by the following formula:

equation image
equation image

where DT is the cell-doubling time, CT is the cell culture time, and PD is the cell-doubling number, Nf is the final number of cells, and Ni is the initial number of cells.

Immunocytochemistry

The hBMSCs in each phase were grown on 12-well plates for 24 hours. Cells were then fixed with 3% paraformaldehyde and permeabilized with Triton X-100. After blocking with 1% BSA, cells were incubated with anti-APE1/Ref-1 antibody (1:250; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) and FITC-conjugated anti-mouse secondary antibody according to the manufacturer's instructions (Santa Cruz Biotechnology Inc.). For characterization of hBMSCs, cells were stained with anti CD54, CD45, CD14, integrin 1-β, and collagen type 1. Slides were coverslipped with mounting medium and photos were taken on an Olympus confocal microscope under ×400 magnification. Mean fluorescence intensity (MFI) was determined by TINA 2.0 program that can relative quantification of fluorescence intensity by nucleus of randomly selected 10 cells.

Analysis of Superoxide Production

Confocal Microscopy

Endogenous superoxide production was evaluated using the oxidative fluorescent dye DHE (dihydroethidium). In each phase of growth of hBMSCs, cells were grown on 12-well plates for 24 hours and then washed with Krebs-HEPES buffer (pH 7.4). Cells were stained with DHE for 15 minutes at 37°C in an incubator. After fixation with paraformaldehyde, slides were coverslipped with mounting medium and photos were taken on an Olympus confocal microscope under ×400 magnification.

Flow Cytometry

The quantification of endogenous superoxide production was done by flow cytometry. Afterward the cells were stained with DHE as above, the cells were removed using trypsin and collected in centrifuge tubes. After centrifugation, pellets were resuspended in Krebs-HEPES buffer (pH 7.4). Fluorescence intensity was calculated for 10,000 cells with Cell Quest software using FACscan (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).

Senescence-Associated β-Galactosidase Assay

Senescence associated β-galactosidase (SA β-gal) was assayed at pH 6.0 as described by Yoon et al. with a slight modification [23]. Briefly, cells were washed twice with PBS, fixed to plates using 3% paraformaldehyde for 3 minutes, washed with PBS, and then incubated overnight in freshly prepared staining in 1 mg/ml 5-bromo-4-chloroindol-3-yl β-D-galactopyranoside (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com, Cat. US10077), 5 mM potassium ferrocyanide, 150 mM NaCl, and 2 mM MgCl2. The stain was visible at 24 hours after incubation at 37°C. The percentage of senescence-associated cells was obtained by counting the number of blue-stained cells and the total cells per field (0.5 × 0.5 cm2) under an inverted microscope. More than 500 cells were counted from five fields and are presented as mean ± SD.

MTT Cell Viability Assay

Viable adherent cells were stained with MTT (3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyl-tetrazolium bromide) (2 mg/ml) for 2 hours. Media were then removed and the formazan crystals produced were dissolved by adding 100 μl of dimethylsulfoxide. Absorbance was assayed at 570 nm and cell viability is expressed as a ratio versus untreated control cells.

Preparation of Cell Lysates and Western Blot Analysis

Proteins were extracted with radioimmunoprecipitation (RIPA) buffer (10 mM TrisHCl, pH 8.0; 150 mM NaCl; 1% Nonidet P-40) containing protease inhibitors (Roche, Mannheim, Germany, http://www.roche.de). Protein concentrations were measured using the Bradford method [24]. Samples were resolved through a 10% SDS-polyacrylamide gel and transferred to Hybond ECL membranes (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). The membrane was blocked in Tris-buffered saline containing 0.1% Tween 20 with 5% nonfat skim milk for 1 hour at room temperature and incubated with primary antibody for 1 hour at room temperature. After three washes in tris-buffered saline containing 0.1% Tween 20 (TBS/T), the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1-hour at reverse transcriptase (RT). After three washes in TBS/T, the membrane was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, U.K.).

RNA Isolation and Reverse Transcriptase Polymerase Chain Reaction

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's instructions. Single-stranded cDNA was synthesized in a 20 μl final volume containing 2 μg of total RNA, 4 μl of 5× Reverse Transcriptase buffer, 4 μl of 2.5 mM dNTP, 1 μl of oligo-dT (100 pmol/μl), 1 μl of RNase inhibitor (4 units/μl), and 1 μl AMV reverse transcriptase (5 units/μl) at 42°C for 1 hour. Afterward the reaction the mixture was boiled for 5 minutes to inactivate RT and quickly chilled on ice. The gene-specific primers used for PCR amplification were: APE1/Ref-1 (Forward: 5′-CGGAATTCCAT GCCGAAGCGTGGGAAAAAGGGAG-3′, Reverse: 5′-CGC AAGCTTTCACAGTGCTAGGTATAGGGTG-3′) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Forward: 5′-AAGGTCGGAGTCAACGGATT-3′, Reverse: 5′-CTC CTGGAAGATGGTGATGG-3′). Ten nanograms of total single-strand cDNA reaction were used as a template. Amplifications were performed in a total volume of 20 μl containing 4 μl of 5× PCR premixture (Elpis Biotech, Daejeon, Korea, http://www.elpisbio.com), in each 1 μl of primer pair (10 pmol/μl). After an initial incubation at 94°C for 5 min, the amplification profile included denaturation at 94°C for 45 seconds, primer annealing at 58°C for 45 minutes, and extension at 72°C for 1 minute. PCR products were analyzed by 1.5% agarose gel-electrophoresis. PCR was performed using a thermal cycler (Mastercycler gradient, Eppendorf, Westbury, NY, http://www.eppendorf.com).

Adenoviral Transfections

An adenovirus encoding green fluorescent protein (AdGFP) and human full length APE1/Ref-1 (AdRef-1) were generated by homologous recombination in HEK 293 cells as previously described [25, 26]. Then, virus-containing media were purified using the Adeno-X virus purification kit purchased from Clontech (Mountain View, CA), according to the manufacturer's instructions. MSCs were infected with the 50 multiplicity of infection (MOI; particle forming units per cell) of adenovirus for 48 hours. We choose the AdGFP as control virus and had consistent results with Clements et al. in transfection efficiency and long-term assessment for hBMSCs [27].

Statistical Analysis

Statistical analyses were done as recommended by an independent analyst. These included the unpaired Student's t test. All values are expressed as mean ± SD, and statistical significance was accepted for p values of <.05.

RESULTS

Determination of the Senescence Phase of hBMSCs: Early, Middle, and Late Phases

BM derived human mesenchymal stem cells (hBMSCs) are somatic cells and achieve a maximum of 30–40 PDs in vitro [28, 29]. During in vitro expansion, broad, enlarged, and flattened morphologic changes were seen from around passage number 9 and progress according to culture time. hBMSCs reached a stationary phase at 35–41 PDs which corresponds to passage number 12 and 13 (Fig. 1A). The results are representative three experiments. The growth curve for hBMSCs suggests that hBMSCs, even if they are stem cells, can be senescent. The Stenderup group divided the life of hBMSCs into three phases according to the time of the life span [2]. We found that the early linear growth curve changed to a hyperbolic curve. To investigate the continuous changes in hBMSCs during senescence, we divided the total life span of hBMSCs into three phases depending on their replication ability. The first is an early phase, before the abrupt increase of one passage time (25 PDs, passage number 7 [p7], 60% of total life span). The second is a middle phase from 26 to 32 PDs (passage numbers 8–10 [p8–10], 60–80% of total life span). The third is a late phase after 32 PDs (passage number 11 [p11], more than 80% of the total life span). For further experiments, we selected p7, p9, and p11 as representatives of early, middle, and late phases, respectively.

To characterize in vitro senescence, hBMSCs were monitored using the SA β-gal assay. Consistent with the growth profile (Fig. 1A), the percent of SA β-gal positive cells was 0% in p7, about 10% in p9, and about 45% in p11 (Fig. 1B-a, 1B-b). Results represent the mean ± SD of three independent experiments (p < .01).

Figure 1.

Long-term growth curve of human bone marrow derived MSCs (hBMSCs) and increases in SA β-galactosidase activity during in vitro expansion. (A): Indicated p7, p9, and p11 represent early, middle, and late phases of the human mesenchymal stem cell life span. Each point represents the mean ± SD. (B-a): To identify senescent cells, hBMSCs were stained for SA β-galactosidase for each passage number. The cells were observed on the indicated day under phase contrast light microscopy and photography. Scale bar = 50 μM. (B-b): Ten percent and 45% staining for SA β-gal at each passage number (p9, p11). p7 did not show staining. Abbreviations: PDs, population doublings; SA β-gal, senescence associated β-galactosidase.

Superoxide Production by hBMSCs During In Vitro Expansion

Recently, it was reported that levels of reactive oxygen species are higher in MSCs obtained from older than younger individuals. However, it is not known whether ROS are involved in the senescence of hMSCs during in vitro expansion. Therefore, we measured superoxide production during in vitro expansion of hBMSCs using DHE (dihydroethidium) staining as described in Materials and Methods section.

As shown in Figure 2A, punctuated red fluorescence was more intense in p11 hBMSCs than p7 hBMSCs, suggesting that superoxide production is closely related with in vitro senescence of hBMSCs. To quantify intracellular superoxide production, DHE stained cells were analyzed by flow cytometry. The p11 hBMSCs showed a right shift compared with p7 hBMSCs (Fig. 2B). In addition, the MFI of p11 hBMSCs increased threefold over p7 hBMSCs that exhibited significantly greater level (p < .01; Fig. 2C).

Figure 2.

Endogenous superoxide increased during mesenchymal stem cell replicative senescence measured by dihydroethidium (DHE) staining and confirmed by FACS analysis. (A): Endogenous superoxide was observed by DHE staining (red) following on confocal microscopy. Scale bar = 20 μM. (B-a): Quantification of endogenous superoxide levels was done by DHE staining followed by fluorescence-activated cell sorting analysis. (B-b): Mean fluorescence intensity was measured along with increases in passage number by fluorescence-activated cell sorting. Each point represents the mean ± SD.

The Expression of APE1/Ref-1 in Each Phase of the Senescence of hBMSCs

Increases in intracellular ROS level induce APE1/Ref-1 expression in a hypoxia/reoxygenation system in endothelial cells or during acute oxidative stress in fibroblasts [16, 30]. As superoxide production increases during the senescence of hBMSCs, we investigated changes in APE1/Ref-1 expression or localization in each phase. In immunofluorescent images, APE1/Ref-1 protein is mainly localized in the nucleus in all phases (Fig. 3A). During the senescence of hBMSCs, intracellular localization of APE1/Ref-1 did not change. However, the fluorescent intensity of APE1/Ref-1 decreased with senescence. Quantitative analysis was performed by measuring the fluorescence intensity in nucleus that is uniformly expressed to fluorescence. MFI decreased about threefold in p11 over p7. Results represent the mean ± SD of three independent experiments (p < .01).

Figure 3.

Redox factor-1 decreases its intracellular level by immunofluorescence, mRNA, and protein change in replicative senescence. (A): Intracellular redox factor-1 was observed by confocal microscopy in each cellular phase. Scale bar = 20 μM. Mean fluorescence intensity was measured in the nucleus of 10 randomly selected cells. (B): Total RNA was isolated from human bone marrow derived MSCs (hBMSCs). For each passage number, the mRNA level was measured by semiquantitative reverse transcriptase polymerase chain reaction. (B, C): Each phase of hBMSCs, RT-PCR, and western blotting detected mRNA and protein levels. Each protein or mRNA expression level was quantified by densitometry. Error bars represent the mean ± SD. Significant difference compared with p7. *p < .05; **p < .01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcriptase polymerase chain reaction.

To evaluate changes in APE1/Ref-1 protein expression with senescence, western blots for APE1/Ref-1 were done using cell homogenates; cells were lysed with RIPA buffer. APE1/Ref-1 protein expression decreased in p9 and p11 compared with p7. The results of western blots were quantified using densitometry. GAPDH was used as a control. APE1/Ref-1 was decreased 20% at p9 and 70% at p11 (Fig. 3B-b) which was statistically significance of three independent experiments. APE1/Ref-1 mRNA levels also decreased, which means that it was downregulated at the transcriptional level (Fig. 3C).

Effect of Exogenous ROS on APE1/Ref-1 Expression in hBMSCs

Besides replicative senescence, there is another model of in vitro senescence – stress-induced senescence [31, 32]. We turned to a hydrogen peroxide induced senescence model to further study hBMSCs senescence. It has not yet been established which concentration of hydrogen peroxide is required to cause growth arrest of hBMSCs. Hence, we examined cell viability with regard to dose and time. A dose of 1 mM hydrogen peroxide had a cytotoxicity such that only 50% of cells were viable after 2 hours. However, a dose of 800 μM hydrogen peroxide had no cytotoxic effect and the percent of viable cells at baseline levels during the entire 48 hours (Fig. 4A). Results represent the mean ± SD of three independent experiments (p < .01).

Figure 4.

Dose-dependent effects of exogenous oxidative stress and decreases in Redox factor-1 mRNA and protein expression during stress induced senescence of human bone marrow derived MSCs (hBMSCs). (A): Cell viability after hydrogen peroxide treatment analyzed by MTT assays. (B): Semiquantitiative RT-PCR of hBMSCs treated with 200 μM, 400 μM, or 800 μM H2O2 for 2 hours and after 48 hours, applied to APE1/Ref-1 and GAPDH primers. (C): Cells were treated with 200, 400, or 800 μM H2O2 for 2 hours and after 48 hours, applied to western blotting for APE1/Ref-1 and GAPDH antibodies. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcriptase polymerase chain reaction.

The APE1/Ref-1 expression level was identified in the H2O2 induced senescence model. APE1/Ref-1 expression also decreased at mRNA and protein levels after treatment with H2O2 for 48 hours (Fig. 4B, 4C). Therefore, we concluded that APE1/Ref-1 expression decreases at the transcription level during in vitro senescence by replication and by oxidative stress.

Change in p21 Expression Level During the Senescence of hBMSCs

p53 can determine cell fate during oxidative stress [13, 33, 34]. If cells are under mild oxidative stress, p53 induces genes for cell cycle arrest and DNA repair. Under extended stress or where there is irreparable DNA damage, p53 induces prooxidant genes to eliminate mutant cells, to maintain cell cycle arrest, and to enter senescence [13]. During replicative senescence, p21 represents one of the downstream elements in the p53 signaling pathway. We thought that p53 and p21 could also regulate APE1/Ref-1 at the transcriptional level during senescence. In western blot analysis, the p53 protein level was slightly decreased at p11. p21 protein was transiently increased at p9. At p11, p21 decreased to less than basal levels (Fig. 5). Therefore, p21 may have an effect on APE1/Ref-1 regulation in the middle phase of in vitro expansion. A reduction in basal p21 expression levels occurs in hBMSCs during the senescent phase by these cells losing their ability to maintain quiescence [11]. Results represent the mean ± SD of three independent experiments (p < .01).

Figure 5.

Change in p21 expression level in each phase of senescence of human bone marrow derived MSCs (hBMSCs). (A): hBMSCs were sampled at the same time in each phase of senescence and analyzed by western blotting using APE1/Ref-1, p53, p21, and GAPDH antibodies. (B, C): Protein expression level was analyzed quantitatively by densitometry. Error bars represent the mean ± SD. Significant difference compared with p7. *p < .05; **p < .01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Overexpression of APE1/Ref-1 Decreases Superoxide Generation and Attenuates Senescence

Chronic treatment of N-acetyl cysteine or supplementation with selenium can reduce senescence markers through reduction of ROS levels [35, 36]. APE1/Ref-1 can also decrease intracellular ROS levels [16]. But these effects have not been reported during senescence.

Compared with mock virus, 50 MOI of Adref-1 overexpression was increased the APE1/Ref-1 expression level to 1.7-fold that was statistically significance in independent three experiments (Fig. 6A). To investigate the effect of APE1/Ref-1 overexpression in senescence, superoxide levels were quantitatively analyzed by fluorescence imaging and FACS with DHE staining of individual of hBMSCs in each phase. In comparison with uninfected cells or mock virus infected cells, cells infected with AdRef-1 showed significant suppression of intracellular superoxide levels (Fig. 6B-a, 6B-b).

Figure 6.

Overexpression of AdRef-1 decreased superoxide accumulation and attenuated SA β-gal. (A): Forty-eight hours after transfection of AdRef-1, overexpression was confirmed by western blotting. (B-a): Endogenous superoxide levels were detected by DHE staining using confocal microscopy. (B-b): Quantification of endogenous superoxide levels was done by DHE staining following fluorescence-activated cell sorting analysis (FACS). (B-c): Mean fluorescence intensity was measured along with increases in passage number by FACS. Each point represents the mean ± SD. (C-a): Overexpression of AdRef-1 changed SA β-gal staining during senescence. (C-b): Statistical analysis of SA β-gal staining showed a significant decrease during the senescence phase compared to mock virus. Significant difference compared with p7. *p < .05; **p < .01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SA β-gal, senescence associated β-galactosidase.

Although the mechanism is not fully understood, generally the SA β-gal assay is believed to be a marker of senescence [37]. The proportion of SA β-gal positive cells was decreased to 50% at p9 and p11 by AdRef-1. Therefore, we conclude that APE1/Ref-1 overexpression attenuates senescence. Results represent the mean ± SD of three independent experiments (p < .01).

DISCUSSION

There are many factors that can cause aging or senescence. Irreversible DNA damage, ROS, and shortening of telomeres are involved. However, we would like to know what the most important cause is and what the initiator of aging is. The ROS theory of aging was among the first [38]. If intracellular ROS levels are not controlled, the increased ROS can cause DNA damage and result in telomere shortening. In this study, we found that hBMSCs senescence is related with APE1/Ref-1, which maintains the intracellular ROS balance.

Differing from embryonic stem cells, adult MSCs undergo senescence. Because MSCs have a role in maintaining tissues and organs, the senescence of MSCs can disrupt the integrity of tissues and organs. Also, recently, MSCs were introduced as cell therapy in irreversible spinal cord injury, and in tendon and ligament regeneration. Therefore, maintenance of natural MSCs characteristics is very important. We confirmed that MSCs in our culture system undergo senescence during in vitro expansion according to a hyperbolic growth curve, and show increased SA β-gal activity and increased stress fibers, these are known as markers of senescence.

The overall balance between oxidants and antioxidants maintains the function of the organ and/or tissue, preventing disease outcome. APE1/Ref-1, one of the Redox modulating genes, expression level decreased during in vitro expansion of hBMSCs despite intracellular ROS level increased. This reduction in APE1/Ref-1 was confirmed in the stress-induced senescence model. These results correlated with the in vivo experiment that APE1/Ref-1 expression reduced in the hippocampus of aged rats [18]. But in case of mild oxidative stress through the low-dose treatment with H2O2, APE1/Ref-1 has been shown to activate [30]. The different expressions of APE1/Ref-1 in each model are seemed to be a result from the degree of strength and the duration of stress to the cell.

There are limited reports on in vitro replicative senescence mechanism of hBMSCs. The glutathione peroxidase deficiency can cause senescence of hBMSCs. It supports the ROS theory of senescence of stem cells [36]. In view of the regulation of oxidative stress by APE1/Ref-1, accelerated senescence could be a result from transcriptional downregulation because the proliferation ability is remained and mRNA level is also decreased. Also, long-term exposure of hBMSCs to ROS leads to senescence. In the stress-induced senescence model, exogenous treatment with H2O2 causes growth arrest and seems to overwhelm intracellular antioxidative capacity. Extended exposure of hBMSCs to oxidative stress reduces APE1/Ref-1 expression levels and can augment the effect of ROS on hBMSCs senescence.

p53 increases its protein level and activity during in vitro senescence, but recently p53 was found to decrease in the last phase of aging [39]. Although p21 is known to be induced by p53, at p7 an increase in p21 was not observed despite a high p53 level. A p21 increase was detected at p9 after APE1/Ref-1 and p53 were decreased. According to studies by Alcorta et al. replicative cellular senescence is a multistep process and p21 expression precedes p16 expression [30]. So p21 is thought to be the initiator of terminal hBMSCs senescence. In the past, APE1/Ref-1 was found to increase p53 transcriptional binding activity. In more recent findings, p53 has role of negatively transcriptional regulation to the APE1/Ref-1 by binding to the promoter region of APE1/Ref-1. We conclude that long lasting increases in p53 activity can induce p21 induction and may suppress APE1/Ref-1 function in hBMSCs, consistent with Zaky's findings [20].

Adenoviral induced APE1/Ref-1 expression in hBMSCs could prevent cellular senescence whereas it reduces SA β-gal staining and intracellular superoxide levels. Although there is no single marker of senescence, SA β-gal staining is believed to be the most reliable marker of senescence. In the SA β-gal assay, APE1/Ref-1 overexpression seemed to reverse senescence but superoxide reduction was observed only at p7, not at p9 or p11 (data not shown). But the reason why there was a difference between phases needs to be further studied.

The ideal protocol for hBMSCs preparation and expansion to improve cell therapy has not yet been defined. It is obvious that APE1/Ref-1 is downregulated during senescence, which means that maintaining a balance between ROS and antioxidants is important in expansion of hBMSCs. In addition, p53 is involved in senescence by regulation of p21 and APE1/Ref-1. Our findings on molecular mechanisms of the limitation of cell expansion may contribute to the prevention of hBMSC senescence and may benefit therapies for otherwise irreparable disease. However, there are some limitations to application to the clinical field. The first, we used adenoviral overexpression system that has a drawback to be introduced into human. The second, we only performed in in-vitro system. Because of some difference between in vitro and in vivo study, the in vivo study must be preceded before it does the application to human. Taken together, clinical application for cell therapy is carefully recommended until the safety is uncovered.

CONCLUSION

We show that the self-renewal ability of MSCs depends on intracellular ROS levels and antioxidative capacity. We obtained novel findings about the relationship between APE1/Ref-1 and MSC senescence. During senescence, extended oxidative stress is a negative transcriptional regulator of APE1/Ref-1 via the p53 pathway. The reduction in APE1/Ref-1 is correlated with the senescence growth curve and increases the speed of development of senescence. By use of an adenoviral overexpression system, APE1/Ref-1 diminishes senescence markers and superoxide accumulation. This could enhance organ and tissue regeneration and can be of benefit in cell therapies.

Acknowledgements

The construct about APE1/Ref-1 adenovirus was kindly donated by T. Finkel (National Institute of Health, USA). This work was supported by the Korea Science & Engineering Foundation through the Infection Signaling Network Research Center (R13-2007-020-01000-0) at Chungnam National University.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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