In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-016-0003-7
Exendin-4 promotes proliferation of adipose-derived stem cells through ERK and JNK signaling pathways Ying Zhang 1 & Shi Chen 2 & Baichuan Liu 3 & Hao Zhou 1 & Shunyin Hu 1 & Ying Zhou 1 & Tianwen Han 1 & Yundai Chen 1
Received: 30 September 2015 / Accepted: 14 January 2016 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2016
Abstract Adipose-derived stem cell (ADSC) transplantation has emerged as a potential tool for the treatment of cardiovascular disease. However, with a limited renewal capacity and the need for mass cells during the engraftment, strategies are needed to enhance ADSC proliferative capacity. In this study, we explored the effects of exendin-4 (Ex-4), a glucagon-like peptide-1 analog, on the growth of ADSCs, focusing in particular on c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. Firstly, ADSCs were isolated and cultured in vitro. Then, flow cytometry demonstrated that ADSCs were positive for CD90 and CD29 but negative for CD31, CD34, and CD45. Ex-4 (0–50 nM) treatment increased ADSC proliferation in a dosedependent manner but had no effects on stem cell markers of ADSCs. Moreover, we found that Ex-4 treatment elevated the phosphorylation levels of the JNK and ERK signaling pathways. Furthermore, utilization of Ex-4 also promoted cyclin D1 and cyclin E protein expression, which was accompanied by more Edu+ cells and a higher percentage of cells in the Sphase of the cell cycle after Ex-4 treatment. In parallel, the application of inhibitors SP600125 and PD98059, inhibitors of the JNK and ERK signaling pathways, respectively, not only reversed such effects of Ex-4 on JNK and ERK but also Ying Zhang, Shi Chen, Baichuan Liu, Hao Zhou and Shunyin Hu contributed equally to this work. * Yundai Chen [email protected]
1
Department of Cardiology, Chinese PLA General Hospital, #28 Fuxing Rd, Beijing 100853, China
2
Department of Radiotherapy, Beijing Cancer Hospital, Beijing, China
3
Department of Emergency, Jishuitan Hospital, Tianjin, China
resulted in lower percentages of S-phase cells and fewer numbers of Edu+ cells. In summary, Ex-4 has no effects on stem cell markers in ADSCs but promotes ADSC growth via JNK and ERK signaling pathways. Keywords Adipose-derived stem cells . Exendin-4 . Proliferation . JNK/ERK signaling pathway
Introduction Acute myocardial infarction (AMI) remains the leading cause of death in humans worldwide, despite advances in medical therapy. AMI results in irrevocable cardiomyocyte damage and cardiomyocyte death, which contributed to the development of heart failure. Adipose-derived stem cells (ADSCs) hold tantalizing potential for the treatment of AMI through self-renewal, differentiating into cardiomyocytes and paracrine cytokines to assist the damaged myocytes (Toma et al. 2002; Tang et al. 2005). Because of these roles, ADSCs are useful in clinical practice for AMI, but it is necessary to isolate sufficient amounts of ADSCs in vitro from adipose tissues. Moreover, maintenance of the stem cell nature of ADSCs during an ex vivo expansion period is also a prerequisite for subsequent transplantation. However, adult stem cells have a limited lifespan compared with embryonic stem cells in vitro, which may restrict the harvesting of a sufficient number of stem cell biomass for an adequate therapy (Wagner et al. 2009). Additionally, after a certain number of cell divisions, ADSCs enter senescence or exhibit a reduced differentiation potential and an absence of stem cell markers (Vacanti et al. 2005). These problems have hindered the expansion of ADSCs for therapeutic uses, causing a major bottleneck in clinical applications.
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Exendin-4 (Ex-4), a glucagon-like peptide-1 analog, is primarily used as an antidiabetic drug for patients with type 2 diabetes (Hausenloy and Yellon 2012). However, in addition to its beneficial metabolic effect, Ex-4 is believed to induce cardioprotective effects, through the activation of ischemiareperfusion injury survival pathways (Lonborg et al. 2012). Recently, researchers found that Ex-4 could promote proliferation and regeneration of β-cells (Li et al. 2005). Moreover, our previous studies have demonstrated that Ex-4 could protect ADSCs against H2O2-induced apoptosis (Liu et al. 2014a; Zhou et al. 2014). However, whether Ex-4 plays a role in the proliferation of ADSCs and the related underlying mechanisms remain unknown. Exposure of cells to Ex-4 activates three main intracellular signal pathways (cyclic adenosine monophosphate [cAMP]/protein kinase A [PKA], phosphatidylinositol 3-kinase [PI3K]/protein kinase B [Akt], and mitogen-associated protein kinase [MAPK]) (Zhu et al. 2014), and MAPK pathways are the major transduction signals that participate in ADSC proliferation (Xu et al. 2012; Sun et al. 2013). Therefore, we postulated that whether Ex-4 could enhance ADSC proliferation in vivo via MAPK pathways. In this study, we investigated the beneficial effects of Ex-4 on ADSC proliferation and determine whether the modulatory role of Ex-4 on ADSCs was due to the activation of MAPK pathways.
Materials and Methods The present study was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of the Chinese PLA (People’s Liberty Army) General Hospital, Beijing, China Isolation and culture characterization of ADSCs ADSCs used in this study were isolated from Sprague-Dawley rats (60–80 g, obtained from the Laboratory Animal Center, Chinese PLA General Hospital, Beijing, China) as described previously (Liu et al. 2014b). Briefly, white fat tissue from the inguinal region was washed with phosphate-buffered saline (PBS) buffer and digested with mixed enzyme (collagenase I (Sigma, St. Louis, MO) and trypsin (Sigma, St. Louis, MO)) at 37°C for 40–45 min with continuous shaking. The acquired tissue was filtered through 70-μm filters and centrifugated for 10 min at 400 g. The obtained cell mass was resuspended in culture medium consisting of L-DMEM (Gibco, Carlsbad, CA) and 10% fetal bovine serum (FBS; HyClone, Logan, UT). The cells were cultured at 37°C/5% CO2, and the medium was replaced every 3 d. The experiments were undertaken with ADSCs in the fourth to fifth passage. For adipogenic differentiation, adipogenic medium was used (DMEM supplemented with 10% FBS, 2 mmol × l−1 of
100 U × ml−1 of penicillin, 100 μg × ml−1 of streptomycin, 100 μmol × l − 1 of L -ascorbate acid, 1 μmol × l−1 of dexamethasone, 0.5 mmol × l−1 of 1-methyl3-isobutylxanthine, and 100 μmol × l−1 of indomethacin) was used. After culturing for 3–4 wks, cells were incubated with Oil Red O solution to stain neutral lipids in the cytoplasm to assess adipogenesis. Osteogenic differentiation was induced by incubating ADSCs at 100% confluence with DMEM supplemented with 10% FBS, 0.1 μmol × l−1 of dexamethasone, 200 μmol × l−1 of L-ascorbic acid, and 10 mmol × l−1 of βglycerol phosphate. After culturing for 3–4 wks, Alizarin Red S was used to assess mineralization and calcium deposits in ADSCs. L-glutamine,
Flow cytometry The immunophenotypes of ADSCs, including assessments of CD29, CD31, CD34, CD45, and CD90 expression, were analyzed by flow cytometry (BD). Briefly, ADSCs in the fourth passage were harvested to detect surface antigens. After being washed twice in PBS, the cells were then incubated with antirat fluorescein isothiocyanate (FITC)-labeled polyclonal antibodies CD29 (1:500; 561796; BD Biosciences), CD31 (1:200; bs-0468R-FITC; Bioss Inc., Woburn, MA), CD34 (1:200; bs-2038R-FITC; Bioss Inc.), CD45 (1:500; 561867; BD Biosciences), and CD90 (1:200; bs0778R-FITC; Bioss Inc.) were added according to manufacture-recommended concentrations. Cells were stained with FITC-labeled IgG to serve as negative controls. To explore whether Ex-4 treatment would influence the immunophenotype of ADSCs, we estimated the marker changes after Ex-4 (1–50 nM) intervention. After treatment, ADSCs were obtained for the detection of CD29, CD31, CD34, CD45, CD90, CD146 (1:200;bs1618R-FITC; Bioss Inc.), and CD271 (1:200;bs-0161RFITC; Bioss Inc.) by flow cytometry based on the above method. Cell viability assay To explore the effects of Ex-4 on ADSCs viability, a 3-(4,5-dimethylthiazohl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) assay was used. Briefly, ADSCs were seeded in 96-well plates with different doses of Ex-4 (0–50 nm/l, SigmaAldrich) for 48 h. After treatment, MTT solution (Sigma-Aldrich) was added into the plates, and the cells were then incubated at 37°C for 4 h. Subsequently, 100 μl dimethyl sulphoxide (DMSO) was added to each well followed by detection of the optical density (OD) value at A490 nm. CCK-8 test For Cell Counting Kit-8 (CCK-8) test, cells were plated onto 96-well plates (1 × 103 cells/well). After treatment with different doses of Ex-4 (0–50 nm/l) in a triplicate pattern,
EXENDIN-4 PROMOTES PROLIFERATION OF CELLS THROUGH SIGNALING
the experiment was continued everyday with the addition of 10 μl of CCK-8 solution for another 2 h at 37°C. The absorbance was determined at a wavelength of 490 nm. The assay was repeated three times. Edu staining assay An 5-ethynyl-2′-deoxyuridine (Edu) staining assay (RIBOBio Co., Guangzhou, China) was performed to observe the proliferative capacity of ADSCs after Ex-4 treatment because Edu is a nucleoside analog of thymidine, which is incorporated into DNA during active DNA synthesis. More Edu+ cells reflect a higher capacity for expansion in vivo. After treatment, ADSCs were incubated with Edu for 2 h then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Then, the Apollo® reaction cocktail (reaction buffer and Apollo® 643 fluorescence) was added into the medium for 30 min in the dark, followed by staining with 4′,6-diamidino-2-phenylindole (DAPI; SigmaAldrich) for 5 min, and the cells were immediately viewed under fluorescence microscopy. The number of Edu+ cells was calculated by counting at least three random separate fields. Cell cycle measurement The DNA content was measured following the staining of the cells with propidium iodide. After indicated treatment, cells were harvested with trypsin, washed once in cold PBS and then fixed in 70% ethanol at −20°C overnight. The fixed cells were pelleted and stained in a propidium iodide solution (50 mg/ml of propidium iodide, 50 mg/ml of RNase A, 0.1% Triton X-100, and 0.1 mM of EDTA) in the dark at 4°C for 1 h prior to flow cytometric quantification of their DNA using a FACScan. Western blotting Total protein extract was obtained by homogenizing ADSCs in RIPA buffer added with a protease inhibitor cocktail. Cell debris was removed by centrifugation, and the supernatant was collected and stored at −80°C. Protein concentrations were determined using a BSA Protein Assay Kit. Total protein extracts were resolved in gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) gels followed and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 1× Tris-buffered saline (TBS) and 0.1% Tween-20 (TBST) with 5% (w/v) bovine serum albumin (BSA) at room temperature for 1 h, followed by an overnight incubation with diluted antibody in blocking buffer at 4°C with gentle shaking. After washing with TBST, the membrane was incubated at room temperature for 1 h with a secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz, CA; 1:2000 dilution). Membranes were visualized with enhanced chemiluminescence followed by exposure to film. The antibodies were p-extracellular signal-regulated kinase (p-ERK; Cell Signaling Technology, Danvers, MA; 1:1000), p-c-Jun NH2terminal kinase (p-c-Jun; Cell Signaling Technology, 1:1000),
cyclin D1 (Santa Cruz, 1:1500), and cyclin E (Santa Cruz, 1:1500). Reagent treatment ADSCs were treated with different concentrations (1, 5, 10, or 50 nM) Ex-4 for 1–7 d, and the surface markers and growth kinetics of ADSCs were then estimated using flow cytometry and a CCK-8 assay, respectively. In some experiments (such as the cell cycle test, Edu staining and WB), ADSCs were pretreated with the ERK signaling pathway inhibitor PD98059 (PD, 10 μm/l, Sigma) or the JNK signaling pathway inhibitor SP600125 (SP, 10 μm/l, Sigma) for 1 h before Ex-4 (10 nm/l) for 48 h. Statistics Data are presented as mean ± SD. Statistical analyses were performed with SPSS software (version 17.0). Statistical significance between two groups was determined by Student’s t test. Comparisons in more than two groups were evaluated by one-way analysis of variance (ANOVA) with a least significant difference test. P < 0.05 was considered as significant difference.
Results Characterization of cultured ADSCs As shown in Fig. 1A, ADSCs cultured in the medium demonstrated fibroblast-like morphology. Importantly, after incubation with adipogenic and osteogenic medium, ADSCs displayed multidifferentiation potential with evidence of showing ADSCs differentiating into adipocytes and osteoblasts. After four passages, most of the ADSCs expressed the mesenchymal stem cell markers CD29 and CD90. By contrast, the ADSCs seldom exhibited expression of the hematopoietic markers CD34 and CD45. The cells were also negative for the expression of the vascular endothelial cell marker CD31. General proliferative capacity of ADSCs First, we measured the general proliferation capacity of ADSCs under normal in vitro conditions with the passage of time. As shown in Fig. 2, after ADSCs in the fourth passage were seeded in culture for approximately 24 h, some parts of the cells attached to the dish and exhibited short, rod-like morphology. Forty-eight hours later, the adherent cells increased gradually and morphologically spread or stretched. Notably, triangle-, polygon-, ellipsoid-, or arc-shaped cells could be observed under microscopic examination. Second, after the cells were cultured for approximately 72 h, we observed an increased number of ADSCs with the typically morphological features that included an enlarged cell body displaying an S-type shape. We also found that the speed of cell proliferation increased during this period and that the majority of cells were similar in morphology. Ninety-six
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Fig. 1 Characterization of ADSCs. (A) Morphology of ADSCs from subcutaneous fat tissue cultured in vitro. Osteogenic and adipogenic differentiation by Alizarin Red S and Oil Red O staining was used to judge ADSC multi-differentiation potential. Oil Red O solution was used to stain neutral lipids in the cytoplasm. Alizarin Red S was used to
assess mineralization and calcium deposits in ADSCs. (B) Surface antigens of ADSCs were characterized by flow cytometry. ADSCs expressed mesenchymal stem cell markers CD29/CD90 but not the hematopoietic marker CD34/CD45 or vascular endothelial cell marker CD31. Bar = 100 μm.
hours later, ADSCs continued to proliferate and divide, exhibiting classical spindle shape and swirl structure. The number of cells increased slightly after 120 h, but there was little cell death, as determined by the appearance of cells floating in the medium. There was hardly any space for cells to grow 144 h later, and most cells showed an oblate or irregular shape, suggesting the saturated growth capacity of the ADSCs.
contribute to the ADSC differentiation, lineage commitment, or senescence of. Thus, we explored the change of the ADSC immunophenotype after Ex-4 incubation by flow cytometry. In Table 2, we found that there was no significant difference in cell surface markers regardless of incubation with or without Ex-4 for 48 h, suggesting that Ex-4 also had no effects on the ADSC immunophenotype.
Effects of Ex-4 on the viability and surface markers of ADSCs To rule out the adverse reaction of Ex-4 on ADSCs, we firstly evaluated the influence of Ex-4 itself on cell viability. As shown in Table 1, at the concentrations used, Ex-4 (1–50 nM) incubation for 48 h had little impact on cell viability compared with normal cells, indicating that Ex-4 had no toxicity effects on ADSCs. Although Ex-4 at the indicated concentrations in our experiment had no influence on ADSCs, we still did not know whether the Ex-4 could alter the stem cell immunophenotype of ADSCs. Because the immunophenotype proteins CD29, CD90, CD146, and CD271 are mesenchymal stem cell markers, if Ex-4 treatment caused a change in the cell surface markers, then it could
Ex-4 improves the proliferation of ADSCs Next, we applied Ex-4 to intervene ADSCs (1–50 nM) for 7 d and then observed the growth of ADSCs treated with Ex-4. Using a CCK-8 assay, we measured the growth kinetics of ADSCs with or without Ex-4. Figure 3 shows that normal ADSCs were initially in a stationary phase during the first 3 d. From the fourth to the sixth day, the cells underwent a logarithmic growth period. However, the cells stayed in the log phase during the following days. Notably, when treated with Ex-4, the cell growth characteristics did not change, but their numbers increased considerably compared with normal cells, indicating that Ex-4 could improve ADSC expansion in a time-dependent manner. Interestingly, no significant
EXENDIN-4 PROMOTES PROLIFERATION OF CELLS THROUGH SIGNALING Fig. 2 General proliferative capacity of ADSCs at different time points. The change of cell numbers of ADSCs under normal condition in vitro with the passage of time was evaluated by an inverted microscope at the indicated time every day for 144 h. About 72–96 h, ADSCs at 72–96 h hold higher proliferative ability than at other time point.
difference in cell expansion was detected during the first 24 h, but a difference between the groups appeared at 48 h. Moreover, we also found that Ex-4-mediated ADSC proliferation was concentration-dependent. With Ex-4 contents in the medium increasing, the number of ADSCs expanded. However, there was no significant difference in cell proliferation ability between the 1 nM group and the normal group. In addition, 10 nM Ex-4 promoted the most ADSC proliferation, albeit the Table 1 Ex-4 had little influence on the viability of ADSCs Control 1 nM Ex-4 5 nM Ex-4 10 nM Ex-4 50 nM Ex-4
OD value
P value
1.15 ± 0.12 1.18 ± 0.10 1.14 ± 0.11 1.15 ± 0.15 1.19 ± 0.11
0.781 0.951 1.000 0.689
number of cells was less than that of the 50 nM group during the first 3 d. Therefore, a 10-nM treatment of Ex-4 for 48 h was used for the following experiments. Roles of the ERK and JNK signaling pathways in Ex-4induced cyclin E/D1 expression Previous studies have demonstrated that Ex-4 is upstream of the activation of MAPK signaling pathways, including ERK, JNK, and P38 signaling pathways. Moreover, ERK and JNK signaling pathways have been proven to participate in ADSC proliferation. Thus, we explored whether Ex-4 increased ADSC growth through ERK and JNK signaling. As shown in Fig. 4A, B, we provided evidence that 10 nM Ex-4 could activate ERK and JNK signaling. Moreover, Ex-4 treatment also could improve the expression of cyclin D1 and cyclin E, key factors that contribute to the transition from G1 to the S-phase in cell cycle. By contrast, blockade of ERK and JNK with pathway inhibitors, not only canceled the effects of Ex-4 on the two pathways but
ZHANG ET AL. Table 2
The change of surface markers of ADSCs with Ex-4 CD29
CD31
CD34
CD45
CD90
CD146
CD271
Control
83.20 ± 0.59
0.35 ± 0.01
0.20 ± 0.02
0.62 ± 0.04
98.86 ± 0.29
66.35 ± 0.72
92.35 ± 0.83
1 nM Ex-4
83.27 ± 0.65 P = 0.896
0.34 ± 0.02 P = 0.694
0.20 ± 0.01 P = 0.859
0.58 ± 0.05 P = 0.377
98.81 ± 0.61 P = 0.875
65.28 ± 0.53 P = 0.622
91.62 ± 0.31 P = 0.493
5 nM Ex-4
83.18 ± 0.72 P = 0.965
0.36 ± 0.03 P = 0.557
0.20 ± 0.02 P = 1.000
0.57 ± 0.04 P = 0.257
98.95 ± 0.42 P = 0.793
63.84 ± 0.75 P = 0.836
90.76 ± 0.61 P = 0.521
10 nM Ex-4 50 nM Ex-4
83.38 ± 0.56 P = 0.741 83.57 ± 0.64 P = 0.490
0.37 ± 0.03 P = 0.335 0.36 ± 0.02 P = 0.557
0.22 ± 0.04 P = 0.485 0.21 ± 0.01 P = 0.723
0.56 ± 0.05 P = 0.127 0.57 ± 0.04 P = 0.257
98.95 ± 0.27 P = 0.785 99.04 ± 0.11 P = 0.588
67.54 ± 0.44 P = 0.323 65.20 ± 0.35 P = 0.431
91.58 ± 0.47 P = 0.653 90.26 ± 0.35 P = 0.343
also eliminated the effects of Ex-4 on cyclin D1/E, suggesting that Ex-4 elevated the expression of cyclin D1/E through ERK and JNK signaling pathways. ERK and JNK pathways involved in the Ex-4-mediated cell cycle transition from G0/G1 to the S-phase Because cell cycle re-entry and the G1/ S transition in proliferative cells commences with the assembly and activation of Cdk4/6-cyclin D and Cdk2-cyclin E (Morgan 1997). Therefore, we proposed that whether Ex-4 promotion of ADSC expansion in vitro was attributed to increases in cyclin D1 and cyclin E that accelerated the transformation from G 0 /G 1 to the S-phase. Thus, we used propidium iodide and Edu to label DNA replication and quantitatively estimated the G0/G1 to the S-phase transition using flow cytometry and Edu+ staining. As shown in Fig. 5A, C, Ex-4 treatment caused the number of cells in the G0/G1 phase to decrease from 90.14% to 68.54%. By contrast, the percentage of cells in the S-phase increased from 7.30% to 21.03% in response to the Ex-4 treatment. To clarify the roles of ERK and
Fig. 3 The growth kinetics of ADSCs with Ex-4 (1–50 nM) for 7 d. CCK-8 test was used to detect the growth kinetics of ADSCs. Cells were plated onto 96-well plates (5 × 103 cells/well) with different doses of Ex-4 (0–50 nm/l) in a triplicate pattern, and the experiment was proceeded everyday by the addition of 10 μl CCK-8 solution for another 2 h at 37°C. The assay was repeated three times. *P < 0.05 vs. control group.
Fig. 4 Ex-4 (10nM) increased the expression of cyclin D1 and cyclin E through the ERK and JNK signaling pathways. (A–D) Ex-4 improved the phosphorylation of ERK and c-Jun. (E–F) Ex-4 regulated the contents of cyclin D1 and cyclin E via ERK and JNK signaling pathways. The expression of cyclin D1, cyclin E, ERK, and c-Jun was evaluated by the Western blots. To establish the role of Ex-4 in the expression of cyclin D1 and cyclin E, ERK signaling pathway inhibitor PD98059 (PD, 10 μm/l, Sigma) or JNK signaling pathway inhibitor SP600125 (SP, 10 μm/l, Sigma) was used for 1 h before Ex-4 (10 nm/l) for 48 h. *P < 0.05 vs. control group; # P < 0.05 VS. PD, SP group, PD: PD98059, SP: SP600125.
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JNK pathways in the Ex-4-mediated cell cycle transition, pathway inhibitors were used. Notably, once the ERK and JNK pathways were blocked, the cells cycle progression was delayed, with evidence of more cells in the G 0 /G l phase and fewer cells in the S-phase. Moreover, Edu staining (Fig. 5B, D) also supported the above results. A higher percentage of Edu+ cells emerged after Ex-4 treatment. However, the inhibition of the ERK and JNK pathways resulted in a lower percentage of Edu + when compared with Ex-4 group. These results indicated that Ex-4 advance the ADSC cell cycle through the ERK and JNK pathways. Fig. 5 Ex-4 improved the proliferative capacity of ADSCs via ERK and JNK signaling pathways which contributed to the cell cycle transition from G0/ G1 to S-phase. (A–C) Ex-4 increased the ratio of Edu+ cells. (B–D) Higher percentage of Sphase cells appeared with Ex-4 treatment. The values on the plots are representative of one experiment. To establish the role of ERK/JNK pathways in the proliferation of ADSCs, PD98059 and SP600125 were used. And the proliferative capacity of ADSCs was detected by flow cytometry and Edu assay. *P < 0.05 vs. control group; # P < 0.05 vs. PD, SP group, PD: PD98059, SP: SP600125.
Discussion Adipose tissue is an alternative source of mesenchymal stem cells and demonstrates an attractive therapeutic potential when transplanted in animal models. There are two phase I clinical trials in progress exploring the effects of ADSC engraftment in the treatment of AMI (Madonna and De Caterina 2010). As is known to all, acquiring a sufficient cell mass of ADSCs for transplantation is a precondition for clinical application. Accordingly, the preparation of large numbers of ADSCs in the laboratory from a relatively small amount of tissue is a difficulty that must be overcome. Meanwhile, maintaining
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ADSCs in an undifferentiated state with no loss of differentiation potential during ex vivo expansion also represents a crucial step. MAPK signaling pathways are the key factors contributing to the survival and growth of many types of cells (Go et al. 2009), The MAPK family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38, the major signal transduction molecules regulated by growth factors, cytokines, and stress (Chang and Karin 2001). Notably, ERK and JNK are activated by phosphorylation and subsequently translocate into the nucleus to promote a variety of gene expression events related to cellular growth (Kim et al. 2011; Zhang et al. 2011; Hsu et al. 2012). Previous studies demonstrated that ERK and JNK signaling pathways were involved in ADSC proliferation in vitro via an improvement in cyclin D1 (Jeon et al. 2006; Baer et al. 2009). Higher cyclin D1 expression results in the formation of the cdk4-cyclin D1 complex and plays a key role in the cell cycle transition from the G0/G1 phase to the S-phases via retinoblastoma protein (Rb) phosphorylation. Moreover, Rb is a critical regulator of the G1-S cell cycle transition because active Rb releases the E2F transcription factors that contributed to the expression of genes implicated in DNA synthesis (Lundberg and Weinberg 1998). Based on this knowledge, many attempts to stimulate the ERK and JNK pathways have been made, but the high cost and adverse side effects have limited their application. In our study, we used a new drug named Ex4, an antidiabetic agent, to treat ADSCs and to observe the mechanism of its effects on ADSC proliferation. We demonstrated that Ex-4 exhibited no toxicity on ADSCs. Furthermore, Ex-4 incubation had no influence on the expression of ADSC stem cell markers of ADSCs, suggesting that Ex-4 may play a role in maintaining the Bstemness^ of ADSCs. Moreover, we found that normal ADSCs displayed a limited expansion capacity in vitro, but Ex-4 intervention could increase ADSC proliferation in a dose- and timedependent manner, and 10 nM of Ex-4 may be the optimal treatment concentration. Furthermore, we found that higher levels of cyclin D1 and cyclin E were present in Ex-4treated ADSCs compared with normal cells. Because an increase in cyclin proteins could contribute to the transformation from the G0/G1 phase to S-phase in cell cycle, we evaluated the cell cycle transition under Ex-4 incubation. Using flow cytometry, we found that the percentage of S-phase cells rose from 7.43 ± 1.31% to 20.93 ± 1.21% after Ex-4 treatment. Moreover, Edu staining was also consistent with this result, showing evidence of more Edu+ cells in the Ex-4 group than in the normal cells. These results indicated that Ex-4 had no effects on its lineage commitment but enhanced ADSCs selfrenewal in vitro through increasing in cyclin D1/E expression. Importantly, we found that JNK and ERK signaling pathways were active after Ex-4 treatment because more p-ERK and pc-Jun proteins were detected in Ex-4-treated cells. Because ERK and JNK are associated with ADSC growth, we
hypothesized that Ex-4 treatment increased the proliferative capacity of ADSCs through ERK and JNK activation. Thus, PD98059 and SP600125, the inhibitors of ERK and JNK signaling pathways, respectively, could be used to block the effects of Ex-4. WB analysis demonstrated that low amounts of cyclin D1/E proteins appeared in Ex-4-treated cells with preinhibition of the ERK and JNK pathways. Moreover, the percentage of S-phase cells in the Ex-4 group also decreased when ERK and JNK were blocked. Edu staining also showed that the number of cells in the division phase decreased once ERK and JNK were inhibited. These data demonstrated that Ex-4 could activate ERK and JNK transduction signals, which enriched both cyclin D1 and cyclin E and accelerated the DNA synthesis. Thus, we illustrated that Ex-4 is important for ADSCs in self-renewal but does not affect ADSC stemness and that ERK and JNK signaling pathways were the main downstream signaling pathways of the effects of Ex-4 on ADSC proliferation. These results provided a new approach to acquire a sufficient cell mass for transplantation without effects on the nature of ADSCs. Previously, many studies have focused on the antidiabetic effects of Ex-4, which include reduced hyperglycemia through the stimulation of the glucose-dependent insulin secretion that contributes to the glucose balance (Tahrani et al. 2011). Recently, Ex-4 was confirmed to be important for cardioprotection via reducing the infarction size, improving the left ventricular ejection fraction (LVEF) (Davidson 2011), and reversing cardiac remodeling (Monji et al. 2013). Our previous studies have found that Ex-4 could protect ADSCs against H2O2-induced apoptosis and could improve the adherence of ADSCs to the cardiomyocytes in vivo (Liu et al. 2014; Zhou et al. 2014). In this study, we demonstrated the effects of Ex-4 on ADSC growth in vitro, showing that Ex-4 could be used as an adjuvant to advance the proliferative capacity of ADSCs. Thus, we offer a new method to acquire sufficient numbers of ADSCs for transplantation in vitro without influencing on its stemness, which may improve the engraftment efficiency. In addition, we identified the ERK and JNK were the main signaling pathways that participate in the Ex-4-mediated ADSC expansion, providing a target for the proliferative capacity of ADSC ex vivo. Acknowledgments The present study was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of the Chinese PLA (People’s Liberty Army) General Hospital, Beijing, China.
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Exendin-4 promotes proliferation of adipose-derived stem cells through ERK and JNK signaling pathways Ying Zhang 1 & Shi Chen 2 & Baichuan Liu 3 & Hao Zhou 1 & Shunyin Hu 1 & Ying Zhou 1 & Tianwen Han 1 & Yundai Chen 1
Received: 30 September 2015 / Accepted: 14 January 2016 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2016
Abstract Adipose-derived stem cell (ADSC) transplantation has emerged as a potential tool for the treatment of cardiovascular disease. However, with a limited renewal capacity and the need for mass cells during the engraftment, strategies are needed to enhance ADSC proliferative capacity. In this study, we explored the effects of exendin-4 (Ex-4), a glucagon-like peptide-1 analog, on the growth of ADSCs, focusing in particular on c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. Firstly, ADSCs were isolated and cultured in vitro. Then, flow cytometry demonstrated that ADSCs were positive for CD90 and CD29 but negative for CD31, CD34, and CD45. Ex-4 (0–50 nM) treatment increased ADSC proliferation in a dosedependent manner but had no effects on stem cell markers of ADSCs. Moreover, we found that Ex-4 treatment elevated the phosphorylation levels of the JNK and ERK signaling pathways. Furthermore, utilization of Ex-4 also promoted cyclin D1 and cyclin E protein expression, which was accompanied by more Edu+ cells and a higher percentage of cells in the Sphase of the cell cycle after Ex-4 treatment. In parallel, the application of inhibitors SP600125 and PD98059, inhibitors of the JNK and ERK signaling pathways, respectively, not only reversed such effects of Ex-4 on JNK and ERK but also Ying Zhang, Shi Chen, Baichuan Liu, Hao Zhou and Shunyin Hu contributed equally to this work. * Yundai Chen [email protected]
1
Department of Cardiology, Chinese PLA General Hospital, #28 Fuxing Rd, Beijing 100853, China
2
Department of Radiotherapy, Beijing Cancer Hospital, Beijing, China
3
Department of Emergency, Jishuitan Hospital, Tianjin, China
resulted in lower percentages of S-phase cells and fewer numbers of Edu+ cells. In summary, Ex-4 has no effects on stem cell markers in ADSCs but promotes ADSC growth via JNK and ERK signaling pathways. Keywords Adipose-derived stem cells . Exendin-4 . Proliferation . JNK/ERK signaling pathway
Introduction Acute myocardial infarction (AMI) remains the leading cause of death in humans worldwide, despite advances in medical therapy. AMI results in irrevocable cardiomyocyte damage and cardiomyocyte death, which contributed to the development of heart failure. Adipose-derived stem cells (ADSCs) hold tantalizing potential for the treatment of AMI through self-renewal, differentiating into cardiomyocytes and paracrine cytokines to assist the damaged myocytes (Toma et al. 2002; Tang et al. 2005). Because of these roles, ADSCs are useful in clinical practice for AMI, but it is necessary to isolate sufficient amounts of ADSCs in vitro from adipose tissues. Moreover, maintenance of the stem cell nature of ADSCs during an ex vivo expansion period is also a prerequisite for subsequent transplantation. However, adult stem cells have a limited lifespan compared with embryonic stem cells in vitro, which may restrict the harvesting of a sufficient number of stem cell biomass for an adequate therapy (Wagner et al. 2009). Additionally, after a certain number of cell divisions, ADSCs enter senescence or exhibit a reduced differentiation potential and an absence of stem cell markers (Vacanti et al. 2005). These problems have hindered the expansion of ADSCs for therapeutic uses, causing a major bottleneck in clinical applications.
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Exendin-4 (Ex-4), a glucagon-like peptide-1 analog, is primarily used as an antidiabetic drug for patients with type 2 diabetes (Hausenloy and Yellon 2012). However, in addition to its beneficial metabolic effect, Ex-4 is believed to induce cardioprotective effects, through the activation of ischemiareperfusion injury survival pathways (Lonborg et al. 2012). Recently, researchers found that Ex-4 could promote proliferation and regeneration of β-cells (Li et al. 2005). Moreover, our previous studies have demonstrated that Ex-4 could protect ADSCs against H2O2-induced apoptosis (Liu et al. 2014a; Zhou et al. 2014). However, whether Ex-4 plays a role in the proliferation of ADSCs and the related underlying mechanisms remain unknown. Exposure of cells to Ex-4 activates three main intracellular signal pathways (cyclic adenosine monophosphate [cAMP]/protein kinase A [PKA], phosphatidylinositol 3-kinase [PI3K]/protein kinase B [Akt], and mitogen-associated protein kinase [MAPK]) (Zhu et al. 2014), and MAPK pathways are the major transduction signals that participate in ADSC proliferation (Xu et al. 2012; Sun et al. 2013). Therefore, we postulated that whether Ex-4 could enhance ADSC proliferation in vivo via MAPK pathways. In this study, we investigated the beneficial effects of Ex-4 on ADSC proliferation and determine whether the modulatory role of Ex-4 on ADSCs was due to the activation of MAPK pathways.
Materials and Methods The present study was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of the Chinese PLA (People’s Liberty Army) General Hospital, Beijing, China Isolation and culture characterization of ADSCs ADSCs used in this study were isolated from Sprague-Dawley rats (60–80 g, obtained from the Laboratory Animal Center, Chinese PLA General Hospital, Beijing, China) as described previously (Liu et al. 2014b). Briefly, white fat tissue from the inguinal region was washed with phosphate-buffered saline (PBS) buffer and digested with mixed enzyme (collagenase I (Sigma, St. Louis, MO) and trypsin (Sigma, St. Louis, MO)) at 37°C for 40–45 min with continuous shaking. The acquired tissue was filtered through 70-μm filters and centrifugated for 10 min at 400 g. The obtained cell mass was resuspended in culture medium consisting of L-DMEM (Gibco, Carlsbad, CA) and 10% fetal bovine serum (FBS; HyClone, Logan, UT). The cells were cultured at 37°C/5% CO2, and the medium was replaced every 3 d. The experiments were undertaken with ADSCs in the fourth to fifth passage. For adipogenic differentiation, adipogenic medium was used (DMEM supplemented with 10% FBS, 2 mmol × l−1 of
100 U × ml−1 of penicillin, 100 μg × ml−1 of streptomycin, 100 μmol × l − 1 of L -ascorbate acid, 1 μmol × l−1 of dexamethasone, 0.5 mmol × l−1 of 1-methyl3-isobutylxanthine, and 100 μmol × l−1 of indomethacin) was used. After culturing for 3–4 wks, cells were incubated with Oil Red O solution to stain neutral lipids in the cytoplasm to assess adipogenesis. Osteogenic differentiation was induced by incubating ADSCs at 100% confluence with DMEM supplemented with 10% FBS, 0.1 μmol × l−1 of dexamethasone, 200 μmol × l−1 of L-ascorbic acid, and 10 mmol × l−1 of βglycerol phosphate. After culturing for 3–4 wks, Alizarin Red S was used to assess mineralization and calcium deposits in ADSCs. L-glutamine,
Flow cytometry The immunophenotypes of ADSCs, including assessments of CD29, CD31, CD34, CD45, and CD90 expression, were analyzed by flow cytometry (BD). Briefly, ADSCs in the fourth passage were harvested to detect surface antigens. After being washed twice in PBS, the cells were then incubated with antirat fluorescein isothiocyanate (FITC)-labeled polyclonal antibodies CD29 (1:500; 561796; BD Biosciences), CD31 (1:200; bs-0468R-FITC; Bioss Inc., Woburn, MA), CD34 (1:200; bs-2038R-FITC; Bioss Inc.), CD45 (1:500; 561867; BD Biosciences), and CD90 (1:200; bs0778R-FITC; Bioss Inc.) were added according to manufacture-recommended concentrations. Cells were stained with FITC-labeled IgG to serve as negative controls. To explore whether Ex-4 treatment would influence the immunophenotype of ADSCs, we estimated the marker changes after Ex-4 (1–50 nM) intervention. After treatment, ADSCs were obtained for the detection of CD29, CD31, CD34, CD45, CD90, CD146 (1:200;bs1618R-FITC; Bioss Inc.), and CD271 (1:200;bs-0161RFITC; Bioss Inc.) by flow cytometry based on the above method. Cell viability assay To explore the effects of Ex-4 on ADSCs viability, a 3-(4,5-dimethylthiazohl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) assay was used. Briefly, ADSCs were seeded in 96-well plates with different doses of Ex-4 (0–50 nm/l, SigmaAldrich) for 48 h. After treatment, MTT solution (Sigma-Aldrich) was added into the plates, and the cells were then incubated at 37°C for 4 h. Subsequently, 100 μl dimethyl sulphoxide (DMSO) was added to each well followed by detection of the optical density (OD) value at A490 nm. CCK-8 test For Cell Counting Kit-8 (CCK-8) test, cells were plated onto 96-well plates (1 × 103 cells/well). After treatment with different doses of Ex-4 (0–50 nm/l) in a triplicate pattern,
EXENDIN-4 PROMOTES PROLIFERATION OF CELLS THROUGH SIGNALING
the experiment was continued everyday with the addition of 10 μl of CCK-8 solution for another 2 h at 37°C. The absorbance was determined at a wavelength of 490 nm. The assay was repeated three times. Edu staining assay An 5-ethynyl-2′-deoxyuridine (Edu) staining assay (RIBOBio Co., Guangzhou, China) was performed to observe the proliferative capacity of ADSCs after Ex-4 treatment because Edu is a nucleoside analog of thymidine, which is incorporated into DNA during active DNA synthesis. More Edu+ cells reflect a higher capacity for expansion in vivo. After treatment, ADSCs were incubated with Edu for 2 h then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Then, the Apollo® reaction cocktail (reaction buffer and Apollo® 643 fluorescence) was added into the medium for 30 min in the dark, followed by staining with 4′,6-diamidino-2-phenylindole (DAPI; SigmaAldrich) for 5 min, and the cells were immediately viewed under fluorescence microscopy. The number of Edu+ cells was calculated by counting at least three random separate fields. Cell cycle measurement The DNA content was measured following the staining of the cells with propidium iodide. After indicated treatment, cells were harvested with trypsin, washed once in cold PBS and then fixed in 70% ethanol at −20°C overnight. The fixed cells were pelleted and stained in a propidium iodide solution (50 mg/ml of propidium iodide, 50 mg/ml of RNase A, 0.1% Triton X-100, and 0.1 mM of EDTA) in the dark at 4°C for 1 h prior to flow cytometric quantification of their DNA using a FACScan. Western blotting Total protein extract was obtained by homogenizing ADSCs in RIPA buffer added with a protease inhibitor cocktail. Cell debris was removed by centrifugation, and the supernatant was collected and stored at −80°C. Protein concentrations were determined using a BSA Protein Assay Kit. Total protein extracts were resolved in gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) gels followed and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 1× Tris-buffered saline (TBS) and 0.1% Tween-20 (TBST) with 5% (w/v) bovine serum albumin (BSA) at room temperature for 1 h, followed by an overnight incubation with diluted antibody in blocking buffer at 4°C with gentle shaking. After washing with TBST, the membrane was incubated at room temperature for 1 h with a secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz, CA; 1:2000 dilution). Membranes were visualized with enhanced chemiluminescence followed by exposure to film. The antibodies were p-extracellular signal-regulated kinase (p-ERK; Cell Signaling Technology, Danvers, MA; 1:1000), p-c-Jun NH2terminal kinase (p-c-Jun; Cell Signaling Technology, 1:1000),
cyclin D1 (Santa Cruz, 1:1500), and cyclin E (Santa Cruz, 1:1500). Reagent treatment ADSCs were treated with different concentrations (1, 5, 10, or 50 nM) Ex-4 for 1–7 d, and the surface markers and growth kinetics of ADSCs were then estimated using flow cytometry and a CCK-8 assay, respectively. In some experiments (such as the cell cycle test, Edu staining and WB), ADSCs were pretreated with the ERK signaling pathway inhibitor PD98059 (PD, 10 μm/l, Sigma) or the JNK signaling pathway inhibitor SP600125 (SP, 10 μm/l, Sigma) for 1 h before Ex-4 (10 nm/l) for 48 h. Statistics Data are presented as mean ± SD. Statistical analyses were performed with SPSS software (version 17.0). Statistical significance between two groups was determined by Student’s t test. Comparisons in more than two groups were evaluated by one-way analysis of variance (ANOVA) with a least significant difference test. P < 0.05 was considered as significant difference.
Results Characterization of cultured ADSCs As shown in Fig. 1A, ADSCs cultured in the medium demonstrated fibroblast-like morphology. Importantly, after incubation with adipogenic and osteogenic medium, ADSCs displayed multidifferentiation potential with evidence of showing ADSCs differentiating into adipocytes and osteoblasts. After four passages, most of the ADSCs expressed the mesenchymal stem cell markers CD29 and CD90. By contrast, the ADSCs seldom exhibited expression of the hematopoietic markers CD34 and CD45. The cells were also negative for the expression of the vascular endothelial cell marker CD31. General proliferative capacity of ADSCs First, we measured the general proliferation capacity of ADSCs under normal in vitro conditions with the passage of time. As shown in Fig. 2, after ADSCs in the fourth passage were seeded in culture for approximately 24 h, some parts of the cells attached to the dish and exhibited short, rod-like morphology. Forty-eight hours later, the adherent cells increased gradually and morphologically spread or stretched. Notably, triangle-, polygon-, ellipsoid-, or arc-shaped cells could be observed under microscopic examination. Second, after the cells were cultured for approximately 72 h, we observed an increased number of ADSCs with the typically morphological features that included an enlarged cell body displaying an S-type shape. We also found that the speed of cell proliferation increased during this period and that the majority of cells were similar in morphology. Ninety-six
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Fig. 1 Characterization of ADSCs. (A) Morphology of ADSCs from subcutaneous fat tissue cultured in vitro. Osteogenic and adipogenic differentiation by Alizarin Red S and Oil Red O staining was used to judge ADSC multi-differentiation potential. Oil Red O solution was used to stain neutral lipids in the cytoplasm. Alizarin Red S was used to
assess mineralization and calcium deposits in ADSCs. (B) Surface antigens of ADSCs were characterized by flow cytometry. ADSCs expressed mesenchymal stem cell markers CD29/CD90 but not the hematopoietic marker CD34/CD45 or vascular endothelial cell marker CD31. Bar = 100 μm.
hours later, ADSCs continued to proliferate and divide, exhibiting classical spindle shape and swirl structure. The number of cells increased slightly after 120 h, but there was little cell death, as determined by the appearance of cells floating in the medium. There was hardly any space for cells to grow 144 h later, and most cells showed an oblate or irregular shape, suggesting the saturated growth capacity of the ADSCs.
contribute to the ADSC differentiation, lineage commitment, or senescence of. Thus, we explored the change of the ADSC immunophenotype after Ex-4 incubation by flow cytometry. In Table 2, we found that there was no significant difference in cell surface markers regardless of incubation with or without Ex-4 for 48 h, suggesting that Ex-4 also had no effects on the ADSC immunophenotype.
Effects of Ex-4 on the viability and surface markers of ADSCs To rule out the adverse reaction of Ex-4 on ADSCs, we firstly evaluated the influence of Ex-4 itself on cell viability. As shown in Table 1, at the concentrations used, Ex-4 (1–50 nM) incubation for 48 h had little impact on cell viability compared with normal cells, indicating that Ex-4 had no toxicity effects on ADSCs. Although Ex-4 at the indicated concentrations in our experiment had no influence on ADSCs, we still did not know whether the Ex-4 could alter the stem cell immunophenotype of ADSCs. Because the immunophenotype proteins CD29, CD90, CD146, and CD271 are mesenchymal stem cell markers, if Ex-4 treatment caused a change in the cell surface markers, then it could
Ex-4 improves the proliferation of ADSCs Next, we applied Ex-4 to intervene ADSCs (1–50 nM) for 7 d and then observed the growth of ADSCs treated with Ex-4. Using a CCK-8 assay, we measured the growth kinetics of ADSCs with or without Ex-4. Figure 3 shows that normal ADSCs were initially in a stationary phase during the first 3 d. From the fourth to the sixth day, the cells underwent a logarithmic growth period. However, the cells stayed in the log phase during the following days. Notably, when treated with Ex-4, the cell growth characteristics did not change, but their numbers increased considerably compared with normal cells, indicating that Ex-4 could improve ADSC expansion in a time-dependent manner. Interestingly, no significant
EXENDIN-4 PROMOTES PROLIFERATION OF CELLS THROUGH SIGNALING Fig. 2 General proliferative capacity of ADSCs at different time points. The change of cell numbers of ADSCs under normal condition in vitro with the passage of time was evaluated by an inverted microscope at the indicated time every day for 144 h. About 72–96 h, ADSCs at 72–96 h hold higher proliferative ability than at other time point.
difference in cell expansion was detected during the first 24 h, but a difference between the groups appeared at 48 h. Moreover, we also found that Ex-4-mediated ADSC proliferation was concentration-dependent. With Ex-4 contents in the medium increasing, the number of ADSCs expanded. However, there was no significant difference in cell proliferation ability between the 1 nM group and the normal group. In addition, 10 nM Ex-4 promoted the most ADSC proliferation, albeit the Table 1 Ex-4 had little influence on the viability of ADSCs Control 1 nM Ex-4 5 nM Ex-4 10 nM Ex-4 50 nM Ex-4
OD value
P value
1.15 ± 0.12 1.18 ± 0.10 1.14 ± 0.11 1.15 ± 0.15 1.19 ± 0.11
0.781 0.951 1.000 0.689
number of cells was less than that of the 50 nM group during the first 3 d. Therefore, a 10-nM treatment of Ex-4 for 48 h was used for the following experiments. Roles of the ERK and JNK signaling pathways in Ex-4induced cyclin E/D1 expression Previous studies have demonstrated that Ex-4 is upstream of the activation of MAPK signaling pathways, including ERK, JNK, and P38 signaling pathways. Moreover, ERK and JNK signaling pathways have been proven to participate in ADSC proliferation. Thus, we explored whether Ex-4 increased ADSC growth through ERK and JNK signaling. As shown in Fig. 4A, B, we provided evidence that 10 nM Ex-4 could activate ERK and JNK signaling. Moreover, Ex-4 treatment also could improve the expression of cyclin D1 and cyclin E, key factors that contribute to the transition from G1 to the S-phase in cell cycle. By contrast, blockade of ERK and JNK with pathway inhibitors, not only canceled the effects of Ex-4 on the two pathways but
ZHANG ET AL. Table 2
The change of surface markers of ADSCs with Ex-4 CD29
CD31
CD34
CD45
CD90
CD146
CD271
Control
83.20 ± 0.59
0.35 ± 0.01
0.20 ± 0.02
0.62 ± 0.04
98.86 ± 0.29
66.35 ± 0.72
92.35 ± 0.83
1 nM Ex-4
83.27 ± 0.65 P = 0.896
0.34 ± 0.02 P = 0.694
0.20 ± 0.01 P = 0.859
0.58 ± 0.05 P = 0.377
98.81 ± 0.61 P = 0.875
65.28 ± 0.53 P = 0.622
91.62 ± 0.31 P = 0.493
5 nM Ex-4
83.18 ± 0.72 P = 0.965
0.36 ± 0.03 P = 0.557
0.20 ± 0.02 P = 1.000
0.57 ± 0.04 P = 0.257
98.95 ± 0.42 P = 0.793
63.84 ± 0.75 P = 0.836
90.76 ± 0.61 P = 0.521
10 nM Ex-4 50 nM Ex-4
83.38 ± 0.56 P = 0.741 83.57 ± 0.64 P = 0.490
0.37 ± 0.03 P = 0.335 0.36 ± 0.02 P = 0.557
0.22 ± 0.04 P = 0.485 0.21 ± 0.01 P = 0.723
0.56 ± 0.05 P = 0.127 0.57 ± 0.04 P = 0.257
98.95 ± 0.27 P = 0.785 99.04 ± 0.11 P = 0.588
67.54 ± 0.44 P = 0.323 65.20 ± 0.35 P = 0.431
91.58 ± 0.47 P = 0.653 90.26 ± 0.35 P = 0.343
also eliminated the effects of Ex-4 on cyclin D1/E, suggesting that Ex-4 elevated the expression of cyclin D1/E through ERK and JNK signaling pathways. ERK and JNK pathways involved in the Ex-4-mediated cell cycle transition from G0/G1 to the S-phase Because cell cycle re-entry and the G1/ S transition in proliferative cells commences with the assembly and activation of Cdk4/6-cyclin D and Cdk2-cyclin E (Morgan 1997). Therefore, we proposed that whether Ex-4 promotion of ADSC expansion in vitro was attributed to increases in cyclin D1 and cyclin E that accelerated the transformation from G 0 /G 1 to the S-phase. Thus, we used propidium iodide and Edu to label DNA replication and quantitatively estimated the G0/G1 to the S-phase transition using flow cytometry and Edu+ staining. As shown in Fig. 5A, C, Ex-4 treatment caused the number of cells in the G0/G1 phase to decrease from 90.14% to 68.54%. By contrast, the percentage of cells in the S-phase increased from 7.30% to 21.03% in response to the Ex-4 treatment. To clarify the roles of ERK and
Fig. 3 The growth kinetics of ADSCs with Ex-4 (1–50 nM) for 7 d. CCK-8 test was used to detect the growth kinetics of ADSCs. Cells were plated onto 96-well plates (5 × 103 cells/well) with different doses of Ex-4 (0–50 nm/l) in a triplicate pattern, and the experiment was proceeded everyday by the addition of 10 μl CCK-8 solution for another 2 h at 37°C. The assay was repeated three times. *P < 0.05 vs. control group.
Fig. 4 Ex-4 (10nM) increased the expression of cyclin D1 and cyclin E through the ERK and JNK signaling pathways. (A–D) Ex-4 improved the phosphorylation of ERK and c-Jun. (E–F) Ex-4 regulated the contents of cyclin D1 and cyclin E via ERK and JNK signaling pathways. The expression of cyclin D1, cyclin E, ERK, and c-Jun was evaluated by the Western blots. To establish the role of Ex-4 in the expression of cyclin D1 and cyclin E, ERK signaling pathway inhibitor PD98059 (PD, 10 μm/l, Sigma) or JNK signaling pathway inhibitor SP600125 (SP, 10 μm/l, Sigma) was used for 1 h before Ex-4 (10 nm/l) for 48 h. *P < 0.05 vs. control group; # P < 0.05 VS. PD, SP group, PD: PD98059, SP: SP600125.
EXENDIN-4 PROMOTES PROLIFERATION OF CELLS THROUGH SIGNALING
JNK pathways in the Ex-4-mediated cell cycle transition, pathway inhibitors were used. Notably, once the ERK and JNK pathways were blocked, the cells cycle progression was delayed, with evidence of more cells in the G 0 /G l phase and fewer cells in the S-phase. Moreover, Edu staining (Fig. 5B, D) also supported the above results. A higher percentage of Edu+ cells emerged after Ex-4 treatment. However, the inhibition of the ERK and JNK pathways resulted in a lower percentage of Edu + when compared with Ex-4 group. These results indicated that Ex-4 advance the ADSC cell cycle through the ERK and JNK pathways. Fig. 5 Ex-4 improved the proliferative capacity of ADSCs via ERK and JNK signaling pathways which contributed to the cell cycle transition from G0/ G1 to S-phase. (A–C) Ex-4 increased the ratio of Edu+ cells. (B–D) Higher percentage of Sphase cells appeared with Ex-4 treatment. The values on the plots are representative of one experiment. To establish the role of ERK/JNK pathways in the proliferation of ADSCs, PD98059 and SP600125 were used. And the proliferative capacity of ADSCs was detected by flow cytometry and Edu assay. *P < 0.05 vs. control group; # P < 0.05 vs. PD, SP group, PD: PD98059, SP: SP600125.
Discussion Adipose tissue is an alternative source of mesenchymal stem cells and demonstrates an attractive therapeutic potential when transplanted in animal models. There are two phase I clinical trials in progress exploring the effects of ADSC engraftment in the treatment of AMI (Madonna and De Caterina 2010). As is known to all, acquiring a sufficient cell mass of ADSCs for transplantation is a precondition for clinical application. Accordingly, the preparation of large numbers of ADSCs in the laboratory from a relatively small amount of tissue is a difficulty that must be overcome. Meanwhile, maintaining
ZHANG ET AL.
ADSCs in an undifferentiated state with no loss of differentiation potential during ex vivo expansion also represents a crucial step. MAPK signaling pathways are the key factors contributing to the survival and growth of many types of cells (Go et al. 2009), The MAPK family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38, the major signal transduction molecules regulated by growth factors, cytokines, and stress (Chang and Karin 2001). Notably, ERK and JNK are activated by phosphorylation and subsequently translocate into the nucleus to promote a variety of gene expression events related to cellular growth (Kim et al. 2011; Zhang et al. 2011; Hsu et al. 2012). Previous studies demonstrated that ERK and JNK signaling pathways were involved in ADSC proliferation in vitro via an improvement in cyclin D1 (Jeon et al. 2006; Baer et al. 2009). Higher cyclin D1 expression results in the formation of the cdk4-cyclin D1 complex and plays a key role in the cell cycle transition from the G0/G1 phase to the S-phases via retinoblastoma protein (Rb) phosphorylation. Moreover, Rb is a critical regulator of the G1-S cell cycle transition because active Rb releases the E2F transcription factors that contributed to the expression of genes implicated in DNA synthesis (Lundberg and Weinberg 1998). Based on this knowledge, many attempts to stimulate the ERK and JNK pathways have been made, but the high cost and adverse side effects have limited their application. In our study, we used a new drug named Ex4, an antidiabetic agent, to treat ADSCs and to observe the mechanism of its effects on ADSC proliferation. We demonstrated that Ex-4 exhibited no toxicity on ADSCs. Furthermore, Ex-4 incubation had no influence on the expression of ADSC stem cell markers of ADSCs, suggesting that Ex-4 may play a role in maintaining the Bstemness^ of ADSCs. Moreover, we found that normal ADSCs displayed a limited expansion capacity in vitro, but Ex-4 intervention could increase ADSC proliferation in a dose- and timedependent manner, and 10 nM of Ex-4 may be the optimal treatment concentration. Furthermore, we found that higher levels of cyclin D1 and cyclin E were present in Ex-4treated ADSCs compared with normal cells. Because an increase in cyclin proteins could contribute to the transformation from the G0/G1 phase to S-phase in cell cycle, we evaluated the cell cycle transition under Ex-4 incubation. Using flow cytometry, we found that the percentage of S-phase cells rose from 7.43 ± 1.31% to 20.93 ± 1.21% after Ex-4 treatment. Moreover, Edu staining was also consistent with this result, showing evidence of more Edu+ cells in the Ex-4 group than in the normal cells. These results indicated that Ex-4 had no effects on its lineage commitment but enhanced ADSCs selfrenewal in vitro through increasing in cyclin D1/E expression. Importantly, we found that JNK and ERK signaling pathways were active after Ex-4 treatment because more p-ERK and pc-Jun proteins were detected in Ex-4-treated cells. Because ERK and JNK are associated with ADSC growth, we
hypothesized that Ex-4 treatment increased the proliferative capacity of ADSCs through ERK and JNK activation. Thus, PD98059 and SP600125, the inhibitors of ERK and JNK signaling pathways, respectively, could be used to block the effects of Ex-4. WB analysis demonstrated that low amounts of cyclin D1/E proteins appeared in Ex-4-treated cells with preinhibition of the ERK and JNK pathways. Moreover, the percentage of S-phase cells in the Ex-4 group also decreased when ERK and JNK were blocked. Edu staining also showed that the number of cells in the division phase decreased once ERK and JNK were inhibited. These data demonstrated that Ex-4 could activate ERK and JNK transduction signals, which enriched both cyclin D1 and cyclin E and accelerated the DNA synthesis. Thus, we illustrated that Ex-4 is important for ADSCs in self-renewal but does not affect ADSC stemness and that ERK and JNK signaling pathways were the main downstream signaling pathways of the effects of Ex-4 on ADSC proliferation. These results provided a new approach to acquire a sufficient cell mass for transplantation without effects on the nature of ADSCs. Previously, many studies have focused on the antidiabetic effects of Ex-4, which include reduced hyperglycemia through the stimulation of the glucose-dependent insulin secretion that contributes to the glucose balance (Tahrani et al. 2011). Recently, Ex-4 was confirmed to be important for cardioprotection via reducing the infarction size, improving the left ventricular ejection fraction (LVEF) (Davidson 2011), and reversing cardiac remodeling (Monji et al. 2013). Our previous studies have found that Ex-4 could protect ADSCs against H2O2-induced apoptosis and could improve the adherence of ADSCs to the cardiomyocytes in vivo (Liu et al. 2014; Zhou et al. 2014). In this study, we demonstrated the effects of Ex-4 on ADSC growth in vitro, showing that Ex-4 could be used as an adjuvant to advance the proliferative capacity of ADSCs. Thus, we offer a new method to acquire sufficient numbers of ADSCs for transplantation in vitro without influencing on its stemness, which may improve the engraftment efficiency. In addition, we identified the ERK and JNK were the main signaling pathways that participate in the Ex-4-mediated ADSC expansion, providing a target for the proliferative capacity of ADSC ex vivo. Acknowledgments The present study was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of the Chinese PLA (People’s Liberty Army) General Hospital, Beijing, China.
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