Article pubs.acs.org/jpr
Low Focal Adhesion Signaling Promotes Ground State Pluripotency of Mouse Embryonic Stem Cells Sara Taleahmad,† Mehdi Mirzaei,‡,§,∥ Azam Samadian,⊥ Seyedeh-Nafiseh Hassani,⊥ Paul A. Haynes,‡ Ghasem Hosseini Salekdeh,*,†,# and Hossein Baharvand*,⊥,¶ †
Department of Molecular Systems Biology and ⊥Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, 1665659911, Iran ‡ Department of Chemistry and Biomolecular Sciences, §Faculty of Medicine and Health Sciences, and ∥Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, 2109 Australia # Department of Systems Biology, Agricultural Biotechnology, Research Institute of Iran, Karaj, Iran ¶ Department of Developmental Biology, University of Science and Culture, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran S Supporting Information *
ABSTRACT: Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state when cultured with 2 inhibitors (2i) of extracellular signal-regulated kinase (MEK) and glycogen synthase kinase-3 (GSK3), and Royan 2 inhibitors (R2i) of FGF4 and TGFβ. The molecular mechanisms that control ESC self-renewal and pluripotency are more important for translating stem cell technologies to clinical applications. In this study, we used the shotgun proteomics technique to compare the proteome of the ground state condition (R2i- and 2i-grown cells) to that of serum. Out of 1749 proteins identified, 171 proteins were differentially expressed (p < 0.05) in the 2i, R2i, and serum samples. Gene ontology (GO) analysis of differentially abundant proteins showed that the focal adhesion signaling pathway significantly downregulated under ground state conditions. mESCs had highly adhesive attachment under the serum condition, whereas in the 2i and R2i culture conditions, a loss of adhesion was observed and the cells were rounded and grew in compact colonies on gelatin. Quantitative RT-PCR showed reduced expression of the integrins family in the 2i and R2i conditions. The serum culture had more prominent phosphorylation of focal adhesion kinase (FAK) compared to 2i and R2i cultures. Activity of the extracellular signal-regulated kinase (ERK)1/2 decreased in the 2i and R2i cultures compared to serum. Activation of integrins by Mn2+ in the 2i and R2i cultures resulted in reduced Nanog and increased the expression of lineage marker genes. In this study, we demonstrated that reduced focal adhesion enabled mESCs to be maintained in an undifferentiated and pluripotent state. KEYWORDS: Pluripotent stem cell, self-renewal, focal adhesion, integrin, extracellular matrix
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signaling pathways, respectively.4 Self-renewal of ESCs is also regulated by interactions that involve cell−cell and cell− extracellular matrix (ECM) contacts and soluble factors.5 Strong adhesive of mESCs culture resulted in cell spreading and decreased pluripotency marker genes.6,7 Comparative proteome analysis of 2i- and serum-grown cells showed that under 2i culture condition, the member of focal adhesion and integrin signaling pathway significantly down-regulated compared to that under the serum condition. 2i-Grown cells were enrichment for proteins associated with glycolysis and gluconeogenesis.8 Soteriou et al. showed that some ECM proteins alone or in combination with fibronectin could act as
INTRODUCTION Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass (ICM) of preimplantation embryos and able to generate three germ layers under differentiated conditions. Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cells (ESCs) is a prerequisite for translation of stem cell technologies to clinical applications.1 Mouse ESCs (mESCs) self-renewal can be maintained in serum-containing medium supplemented with leukemia inhibitor factor (LIF) through the activation of signal transducer and activator of transcription 3 (Stat3),2 or cultured under 2 inhibitors (2i), PD0325901 and CHIR99021, selectively targeting mitogen-activated protein kinase (MEK) and glycogen synthase kinase-3 (GSK3),3 or Royan 2 inhibitors (R2i) PD0325901 and SB431542, inhibiting FGF4 and TGFβ © 2017 American Chemical Society
Received: May 22, 2017 Published: August 29, 2017 3585
DOI: 10.1021/acs.jproteome.7b00322 J. Proteome Res. 2017, 16, 3585−3595
Article
Journal of Proteome Research Trypsin In-Gel Digestion
substrate to regulate hESCs self-renewal. Therefore, the selfrenewal of stem cells could be related to the balance between molecular composition and ECM network properties.9 ECM signaling is mediated largely by the integrin family of cell surface adhesion receptors that consists of α and β subunits. At the cellular level, integrins play an important role in cell migration, spreading, proliferation, survival, morphogenesis, and gene expression.10,11 In particular, focal adhesion kinase (FAK) and Src play central roles in integrin-mediated signaling cascades.12 Aggregation of FAK with integrins and cytoskeletal proteins in focal contacts has been proposed to be responsible for FAK activation and autophosphorylation by integrins in cell adhesion. The activated FAK forms a complex with Src family kinases, which initiates multiple downstream signaling pathways that regulate different cellular functions.13 Here, by using a shotgun proteomics approach, we have shown that reduction of the ECM−integrin interaction under 2i and R2i conditions plays a role in the maintenance of mESC pluripotency. Activation of ECM−integrin interaction by Mn2+, a strong activator of integrin function, leads to phosphorylation of FAK and inhibits mESC self-renewal by reduction of pluripotency marker gene expressions.
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Each stained gel lane was cut into 12 equal size pieces. Each piece was further cut and placed into wells of a 96-well plate. To destain, the gel pieces were briefly washed with 100 mM NH4HCO3, twice washed with 200 μL of ACN (50%)/100 mM NH4HCO3 (50%) for 10 min, and finally dehydrated with 100% ACN. The samples were air-dried and reduced with 50 μL of 10 mM DTT/NH4HCO3 (50 mM) at 37 °C for 1 h before alkylating in the dark with 50 μL of 50 mM iodoacetamide/NH4HCO3 (50 mM) at room temperature for 1 h. The samples were briefly washed with 100 mM NH4HCO3, 200 μL of ACN (50%)/100 mM NH4HCO3 (50%) for 10 min, dehydrated with 100% ACN, and then airdried. Finally, samples were digested with 20 μL of trypsin (12.5 ng/mL of 50 mM NH4HCO3) for 30 min on ice, and then incubated overnight at 37 °C. Peptides that resulted from the trypsin digestion of the proteins were extracted twice with 30 μL of ACN (50%)/formic-acid (2%), dried, vacuum centrifuged, and reconstituted to 10 μL with 2% formic acid. Nanoflow Liquid Chromatography-Tandem Mass Spectrometry
The tryptic digest extracts from 1 DE gel slices were analyzed by nanoLC-MS/MS using a LTQ-XL ion-trap mass spectrometer (Thermo, Fremont, CA). Reverse phase columns were packed in-house to approximately 7 cm (100 μm i.d.) using 100-Å, 5-μM Zorbax C18 resin (Agilent Technologies, Santa Clara, CA) in a fused silica capillary with an integrated electrospray tip. A 1.8-kV electrospray voltage was applied via a liquid junction upstream of the C18 column. Samples were injected onto the C18 column using a surveyor autosampler (Thermo, Fremont, CA). Each sample was loaded onto the C18 column followed by an initial wash step with buffer A (5% (v/v) ACN, 0.1% (v/v) formic acid) for 10 min at 1 μL/min. Peptides were subsequently eluted from the C18 column with 0%−50% buffer B (95% (v/v) ACN, 0.1% (v/v) formic acid) over 58 min at 500 nL/min followed by 50%−95% buffer B over 5 min at 500 nL/min. The column eluate was directed into a nanospray ionization source of the mass spectrometer. Spectra were scanned over the range of 400−1500 amu. Automated peak recognition, dynamic exculsion window set to 90s14 tandem MS of the top six most intense precursor ions at 35% normalization collision energy were performed using Xcalibur software (version 2.06) (Thermo, Fremont, CA).
MATERIALS AND METHODS
Culture of Mouse Embryonic Stem Cells (mESCs)
In this study, we used a previously established mESC line, Royan B20. Three biological repeats of the cells were cultured on 0.1% gelatin-coated plates (Sigma-Aldrich) and passaged 8 times in R2i/leukemia inhibitory factor (LIF) and 2i/LIF (serum-free N2B27 medium) and serum/LIF medium. The 2i treatment included MEK and GSK3 inhibitors PD0325901 (1 μM; Stemgent) and CHIR99021 (3 μM; Stemgent).3 R2i culture contained 1 μM PD0329501 and 10 μM SB431542 which inhibited the TGFβ signaling pathway.4 Serum medium consisted of knockout Dulbecco’s modified Eagle’s medium (KoDMEM; Invitrogen), 15% fetal bovine serum (FBS; HyClone), 1% nonessential amino acids, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.1 mM βmercaptoethanol. N2B27/LIF medium contained a 1:1 ratio of DMEM/F12 (Invitrogen) and neurobasal (Invitrogen), 1% B27 supplement (Invitrogen), 1% N2 supplement (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 2 mM L-glutamine (Invitrogen), 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen), and 5 mg/mL bovine serum albumin (BSA; Sigma-Aldrich). Embryoid bodies were produced from mESCs using nonadherent bacteriological plastic dishes in the presence of complete growth medium lacking leukemia inhibitory factor (LIF) containing 10% (v/v) FBS for 6 days.
Protein Identification
The resultant raw files were converted to mzXML format and processed through free software from the Global Proteome Machine (GPM) using version 2.1.1. of the X! Tandem algorithm, obtained from http://www.thegpm.org.15 For each experiment, we processed the 12 fractions sequentially with output files for each individual fraction. A merged, nonredundant output file was generated for protein identifications with log (e) values less than −1. Tandem mass spectra were searched against the NCBI O. Search parameters included MS and MS/MS tolerances of ±2 Da and ±0.2 Da, tolerance of 2 missed tryptic cleavages, and K/R-P cleavages. Fixed modifications were set for carbamidomethylation of cysteine, and variable modifications were set for oxidation of methionine.
Protein Extraction and Separation by Sodium Dodecyl Sulfate (SDS)
The 2i, R2i, and serum-grown cells (at least 5 × 106 cells) were collected and washed twice with 5 mL of ice-cold PBS. The samples were then centrifuged at 450g for 5 min at 4 °C. After discarding the supernatant, the plated cells were frozen at −80 °C until analysis. Sample preparation for mass spectrometry that included protein extraction and sodium dodecyl sulfate (SDS)-PAGE separation were performed as previously described.8
Quantitative Proteomic Analysis
Protein abundance data were calculated based on normalized spectral abundance factors (NSAF) as described previously.16 For each protein k, in sample i, the number of spectral counts 3586
DOI: 10.1021/acs.jproteome.7b00322 J. Proteome Res. 2017, 16, 3585−3595
Article
Journal of Proteome Research
Figure 1. K-mean clustering and GO analysis of differentially expressed proteins. These proteins could be clustered into seven different groups. Clusters 1−4 represented up-regulated proteins and clusters 5−7 represented down-regulated proteins. This list of proteins was employed to identify significant biological processes by comparing their functional annotations according to the PANTHER classification system. Proteins involved in the metabolic process were enriched in up-regulated protein clusters (clusters 1−4), whereas the down-regulated proteins in clusters 5, 6, and 7 enriched in the developmental process.
the results of each comparison test (up- and down-regulated proteins). The lists of up- and down-regulated proteins with relative gene identifiers for each comparison were uploaded in Database for Annotation, Visualization and Integrated Discovery (DAVID). The p-values (
