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Overexpression of the 14-3-3γ protein in uterine leiomyoma cells results in growth retardation and increased apoptosis
Cellular Signalling 45 (2018) 43–53
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Overexpression of the 14-3-3γ protein in uterine leiomyoma cells results in growth retardation and increased apoptosis ⁎
Qi Shena, Xiaoli Hua, Lulu Zhoua, Shuangwei Zoua, Lu-Zhe Sunb, , Xueqiong Zhua, a b
T
⁎
Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China Department of Cell Systems & Anatomy, University of Texas Health Science Center at San Antonio, TX, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Uterine leiomyoma 14-3-3γ Proliferation Apoptosis Bioinformatics Signaling pathway
Protein 14-3-3γ was significantly reduced in human uterine leiomyoma compared to the adjacent normal myometrium tissue. To investigate the possible link between the reduced 14-3-3γ expression and uterine leiomyoma growth, we have overexpressed 14-3-3γ protein in uterine leiomyomal cells and its effects on cell proliferation and apoptosis were analyzed. Over-expression of 14-3-3γ was achieved by transducing into two types of uterine leiomyoma cells (primary culture cells and immortal stem cells) with a 14-3-3γ expressing adenovirus vector. Differentially expressed proteins were screened by the proteomics tool (TMT-LCTMS), followed by PANTHER database analysis to single out specifically modified signaling pathway proteins, which were confirmed by Phospho-MAPK Antibody Array and Western blots analysis. The results showed that increase in 143-3γ expression in both two types of human uterine leiomyoma cells inhibited cell proliferation and induced apoptosis. Proteomic screening has found 42 proteins, among 5846, that were significantly affected. PANTHER database and GeneMANIA analysis of the differentially expressed proteins have found that proteins involved in apoptosis signaling and cytoskeletal/adhesion were among the ones affected the most. Further analysis of the key signaling pathways have found that over-expression of 14-3-3γ resulted in reductions in the phosphorylations of multiple signaling molecules, including AKT, pan, ERK1/2, GSK-3 α/β, MEK1/2, Foxo1 and Vimentin. In conclusion, the loss of 14-3-3γ may have causal effects on the growth of uterine leiomyoma, which may function through modifying multiple signaling pathways, including AKT-Foxo and/or MEK1/2-ERK1/2.
1. Introduction Uterine leiomyomas are among the most common tumors in women, affecting 20% to 50% of reproductive women, which can cause menorrhagia, abnormal uterine bleeding, pelvic pain, infertility and miscarriage [1]. Except invasive surgical procedure, there is no simple, safe and effective way to treat the disease currently. Uterine leiomyomas are generally considered as a hormonal dependent tumor, closely related to estrogen and progesterone, but the mechanism of tumorigenesis is still unclear [2]. In our previous study, we have found that 14-3-3γ exhibited a marked down-regulation in leiomyoma tissues compared with the adjacent normal myometrium, via proteomic and reverse transcription polymerase chain reaction (RT-PCR) techniques [3]. In this study, we have tested the hypothesis that the loss of 14-3-3γ protein may play a causal role in the origin or growth of leiomyoma.
14-3-3γ is a member of 14-3-3 proteins family (7 isoforms: β, γ, ε, η, σ, τ/θ and ξ), a highly conserved phosphoserine/threonine-binding proteins group that regulates diverse cellular processes, including cell cycle progression, transcriptional regulation, apoptosis, and cell proliferation [4,5]. 14-3-3 proteins have been found to play roles in human tumorigenesis, with different subtypes expressed in different cancer tissues and possible different regulatory mechanisms involved [6]. At present, 14-3-3γ has been reported in breast cancer, lung cancer and glioma [7–9]. However, the role of 14-3-3γ in gynecological tumors has not been reported. Mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signal transduction pathways are capable of regulating the proliferation and apoptosis of uterine leiomyoma cells, which are considered to be the main signal pathways that are responsible for the development of uterine leiomyomas [10,11]. In
Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; MAPK, mitogen-activated protein kinases; PI3K/AKT, and phosphatidylinositol 3-kinase/protein kinase B; ERK1/2, extracellular regulated protein kinases 1/2; TMT, tandem mass tag; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; CCK-8, cell counting kit-8; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel; LSD, least significance difference ⁎ Corresponding authors at: No. 109 Xueyuan Xi Road, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China. E-mail addresses: [email protected] (L.-Z. Sun), [email protected] (X. Zhu). https://doi.org/10.1016/j.cellsig.2018.01.025 Received 17 December 2017; Received in revised form 22 January 2018; Accepted 25 January 2018 0898-6568/ © 2018 Published by Elsevier Inc.
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culture medium above, under 2.5% O2 and 5% CO2 at 37 °C. Culture medium was changed every other day. Cells were split as 1:2 every 4 to 6 days. Cells from third passages to the fifth were used for the experiments.
uterine leiomyoma cells, for example, estrogen is capable of increasing cell proliferation via protein kinase C, which further activates the extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway. Interestingly, in normal myometrium cells, estrogen is also capable of activating protein kinase C, but instead of stimulating MAPK/ERK1/2 signaling, the kinase inactivates MAPK/ERK1/2 pathway, which resulted in low level of cell proliferation activities. The difference in the signaling transductions of the two cells/systems is still unclear. Progesterone induces PI3K/AKT pathway to activate the downstream genes (GSK3, TSC2, Foxo, and others), which can inhibit the leiomyoma cells apoptosis and also promote cell proliferation [11]. 14-3-3γ is a binding partner of both downstream members of MAPK (such as JNK, p38 and ERK1/2) and PI3K/AKT (such as Foxo, BAD and BAX), playing an important role in cell proliferation and apoptosis via preventing the members from dephosphorylation, which further controls the proportion of cytoplasmic and nuclear protein distributions [12]. In this study, we tested a hypothesis that loss of 14-3-3γ by uterine leiomyoma cells plays a causal role in the development of leiomyoma by modifying MAPK and/or PI3K/AKT signaling pathways. Tandem mass tag (TMT) is a new proteomics technology that can quantify the absolute amount of proteins by specifically labeling the peptides and proteins [13,14]. Compared to the traditional bidirectional gel electrophoresis proteomics, TMT has clear advantages in detection range, reproducibility, high flux, accuracy and high resolution [15]. Since its invention, TMT has been used widely in cancer researches, such as breast-, liver-, and colorectal-cancers and others [16–18]. In this study, TMT was used to identify differently expressed proteins in cells after 14-3-3γ up-regulated, phospho-MAPK antibody array and Western blots were used for further conformation. Overall, our study tried to explore the role of 14-3-3γ in leiomyoma cell proliferation and apoptosis, and related signal pathways, which will help us better learn the biological effects of 14-3-3γ, and also provide experimental basis for clinical treatment on uterine leiomyoma patients.
2.3. Staining of α-smooth muscle actin by immunocytochemistry Uterine leiomyoma cells were identified by the expression of αsmooth muscle actin. Cells were fixed with 4% paraformaldehyde, washed and then permeabilized in PBS containing 0.2% Triton X-100 for 15 min. Cells were then incubated in a serum-free blocking solution for 15 min at room temperature, incubated with mouse monoclonal anti-α-smooth muscle actin antibody (1:100 dilution; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) overnight at 4 °C. After extensive washing with PBS, cells were incubated with biotinylated goat anti-mouse IgG as secondary antibody. After incubation the bound antibodies were visualized using 3,3′-diaminobenzidine. Finally, nuclei were stained with hematoxylin. Cells incubated with PBS, instead of primary antibody, was included as negative control. 2.4. Construction and characterization of recombinant plasmid vectors Human 14-3-3γ (Genebank: NM_012479.3) gene was synthesized by GenScript company (Shanghai, China). The vector plasmid (pHBAdMCMV-RFP, Hanbio, Shanghai, China) was digested by the BamHI and EcoRI restrictive enzymes, and purified using agarose gel electrophoresis. Then 14-3-3γ gene was subcloned into the pHBAd-MCMV-RFP. The positive recombinant clone, named pHBAd-MCMV-RFP-14-3-3γ, was then transfected and amplified into DH5α by hot shock method and screened by Amp + resistance. The positive clones were finally identified and characterized by PCR and sequencing. 2.5. Virus preparation, purification, and titer determination The pHBAd-MCMV-RFP-14-3-3γ and the adenovirus helper plasmid pHBAd-BHG (Hanbio, Shanghai, China) were co-transformed into HEK293 cells with lipofectamine 2000 Kit according to the manufacturer's instructions. Six hours after transformation, the medium was replaced with new DMEM, and cytopathic effects were observed every 2 days. The viral particles in the HEK293 cells were prepared by lysis of the cells with three consecutive freeze–thawing cycles in ice-ethanol, then by passing through 0.4 μM syringe filter to remove inadvertent materials. The viral titer (pHBAd-MCMV-RFP-14-3-3γ) was detected by the TCID50 standard method. Finally, the recombinant adenovirus was stored at −80 °C until use. pHBAd-MCMV-RFP-14-3-3γ virus was used to over-express 14-3-3γ proteins, while pHBAd-MCMV-RFP virus was used as a negative control.
2. Materials and methods 2.1. Primary tissue collection and stem cell Uterine leiomyoma tissues were obtained from 40 patients with ages ranged 43 ± 6 years, between October 2013 and July 2015. The tissues were collected during the procedures of uterine myomectomy, subtotal or total hysterectomy. Patients with following characters were excluded: administrations with medicines or hormones within 3 months prior surgery, or with other complications, such as infections, chronic diseases (such as diabetes and hypertension), uterine malignancy and/ or adenomyosis (on the basis of tissue pathology). The study protocol was approved by the Research Ethical Committee of the Second Affiliated Hospital of Wenzhou Medical University and met the standards of the Declaration of Helsinki. Written consent was obtained from every patient before the procedures. Human uterine leiomyoma stem cells were from Dr. Ayman AlHendy Lab, we have immortalized them with hTERT lentivirus.
2.6. Cell counting kit-8 (CCK-8) assay To detect the cell proliferation, 1 × 104 cells/well were seeded into 96-well plates and incubated at 37 °C with 5% CO2. After 24 h of incubation, the cells were treated with 14-3-3γ over-expression virus or control virus. By end of treatments, 10 μl of CCK-8 (Dojindo, Kumamoto, Japan) was added to each well and incubated for an additional 2 h. The reaction product was quantified by spectrophotometry at 450 nm wavelength, and the percentage of viability or number of cells were calculated by formula: (treated cells absorbent / non treated cells absorbent) × 100.
2.2. Primary human cell and stem cell culture Fresh tissues were washed with cold phosphate-buffered saline (PBS), then cut into small pieces (1 mm3) and digested with 0.2% (v/v) collagenase II (Invitrogen, Carlsbad, CA, USA) in Dulbecco's modified Eagle's medium (DMEM) for 4 h in a 37 °C with shaking. The dissociated cells were centrifuged at 400 ×g for 5 min. The resultant cell deposit was suspended with complete culture medium (DMEM, 10% fetal bovine serum, 100 IU/ml of penicillin G and 100 μg/ml streptomycin) and centrifuged at 400 ×g for 5 min. The resultant cells were cultured at a density of 2 × 105 cells/ml under 5% CO2 at 37 °C in the complete culture medium. Stem cells were cultured with the same complete
2.7. Flow cytometric assay of apoptosis with APC/7-AAD staining Cell apoptosis was assayed by flow cytometry after APC/7-AAD staining. APC was used to detect early apoptosis in cellular membrane, 7-AAD was used to detect late apoptosis in cell nucleus. The assay was performed according to the manufacturer's guidelines. Briefly, after 44
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biotinylated antibodies. Finally the membranes were incubated with streptavidin horseradish peroxidase-conjugated antibody. Immunoreactivity was visualized using a chemiluminescent substrate. Densitometric analysis was performed using GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, USA).
washed twice with cold PBS and then re-suspended in 1× binding buffer, cells (105 cells in 100 μl of the solution) were mixed with 5 μl of APC and 5 μl 7-AAD. After gently vortexed, the cells were incubated for 15 min at room temperature (25 °C) in the dark and then were added 200 μl of 1× binding buffer to each tube, and then analyzed by flow cytometry within 1 h.
2.11. TMT combined with liquid chromatography-tandem mass spectrometry
2.8. Apoptosis assay by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining
Cells were lysed in Radio-Immunoprecipitation Assay lysis buffer and sonicated. The protein lysates were denatured, reduced, and alkylated as recommended by the TMT Mass Tagging Kit (Thermo Fisher Scientific, Waltham, MA, USA) protocol. Trypsin was added at a protein/enzyme ratio of 30:1 by mass and the digestion was performed at 37 °C overnight. Peptides were labeled with TMT reagents according to the manufacturer's protocol in duplicate and equal amounts of labeled peptides were combined to obtain one sample, which was separated into 9 fractions by strong cation exchange chromatography using TopTip columns (PolyLC). Elution was performed with increasing concentrations of KCl (from 0 to 0.5 M). Eluates were dried using a SpeedVac and then desalted using C18/hypercarb PolyLC. Samples from each fraction were run in triplicate on an Orbitrap Pro mass spectrometer that was coupled to a nanoLC (Thermo Fisher Scientific), and the spectra obtained were analyzed with Proteome Discoverer 1.3 software against the Human International Protein Index (IPI) database. Peptides were identified with a false discovery rate of < 1%.
For the analysis of cell death, apoptotic cells were labeled with TUNEL staining (Roche, Indianapolis, IN, USA). TUNEL was used to detect apoptosis in cell nucleus. The procedure was done according to the manufacturer's instruction. The apoptotic cells were identified by nuclear staining of dark brown colors, which was quantified by counting 100 cells from six random microscopic fields in each group, and the values were presented as the percentage of positive cells in the total number of cells. 2.9. Cell lysate preparation and Western blot analysis Total proteins were prepared by whole-cell lysis with the buffer (60 mM Tris-HCl, pH 6.8, 5% glycerol, 2% SDS) on ice. Cytoplasmic and nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, IL, USA) according to the manufacture's protocol. The protein from each experimental group was quantified by bicinchoninic acid method (Beyotime, Jiangsu, China). Cellular proteins (30 μg) were solubilized in sample buffer (4% SDS, 30 mm dithiothreitol, 0.25 m sucrose, 0.01 m EDTA-Na2 and 0.075% bromophenol blue), and heated at 100 °C for 5 min to denature proteins. The proteins were separated using electrophoresis on 12% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) and then electro-transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 2 h at room temperature in 0.05 M Tris-buffered saline with 0.5% triton X-100 (TBS-T, pH 7.4) containing 5% skim milk and then incubated in TBS-T overnight at 4 °C with one of the appropriate primary antibodies at 1:1000 dilutions, unless specified otherwise. The primary antibodies used were 14-3-3γ (Santa Cruz Biotechnology, Santa Cruz, CA, USA); AKT (CST, Boston, USA); p-AKT (CST, USA); Foxo1 (CST, USA); p-Foxo1 (CST, USA); ERK1/2 (CST, USA); p-ERK1/2 (CST, USA); MEK1/2 (CST, USA); p-MEK1/2 (CST, USA); Vimentin (Abways, Shanghai, China); p-Vimentin (Abways, China); TSC2 (CST, USA); p-TSC2 (CST, USA); mTOR (Abways, China); p-mTOR (Abways, China); TGFBI (Abways, China); Desmin (Abways, China); Lamin B (Bioworld, St. Louis Park, MN, USA); β-Actin (1: 2000 dilution; Beyotime, China); and α-Tubulin (1:2000 dilution; Beyotime, China). After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 for anti-rabbit-IgG or anti-mouse-IgG) for 1 h at room temperature. Blots then were developed by an enhanced chemiluminescence. The protein bands were relatively quantified by densitometry scanning using the AlphaEaseFC 4.0 software (Alpha Innotech Corp, San Leandro, CA, USA), then normalized by corresponding levels of α-Tubulin or β-Actin respectively. Each experiment was repeated at least three times.
2.12. Bioinformatic analysis Biological processes, molecular functions and the pathways of the differentially expressed proteins involved were analyzed by PANTHER (Protein annotation through evolutionary relationship classification system; http://www.pantherdb.org/) consortium databases [19]. The combination of gene set enrichment analysis and network analysis were analyzed by GeneMANIA [20]. 2.13. Statistical methods All statistical analyses were performed with SPSS17.0 software. Data was presented as the mean ± standard deviation. Difference between two groups was analyzed by Student's t-test. Differences among multiple groups were analyzed by one-way ANOVA. If variances were homogeneous, then least significance difference (LSD) method was used to compare between two groups. If variances were nonhomogeneous, Dunnett's T3 method was applied to compare between two groups. A 2tailed P value < 0.05 was considered statistically significant. 3. Results 3.1. Culture of human uterine leiomyoma cells and infection with 14-3-3γ adenovirus vectors Human uterine leiomyoma cells were isolated from uterine leiomyoma tissues of patients aged 43 ± 6 years-old by collagenase digestion and step-wise centrifugations. A uniformed population with smooth muscle cell characteristics was achieved after 2–3 passages. To confirm the purity of the cells, immunocytochemistry staining of the cells with α-actin antibody was performed. As shown in Fig. 1(A, B), the positive staining of this smooth muscle-specific actin indicated that almost 100% of cells derived from uterine leiomyoma tissues retained their smooth muscle characteristics. To generate a virus vector expressing human 14-3-3γ gene, pHBAdMCMV-RFP plasmids were digested by the BamHI and EcoRI, then 14-33γ was subcloned into the vector. The positive recombinant clones were selected, confirmed by PCR and sequencing (Fig. 1C, D, E). The uterine leiomyoma cells at the passage 3 were infected with 14-3-3γ
2.10. Human phospho-MAPK antibody array MAPK protein phosphorylation was screened with 300 μg of cell extracts using Human Phospho-MAPK Array Kit according to the manufacturer's instructions (Proteome Profiler; R&D Systems, Minneapolis, MN, USA). Human Phospho-MAPK Array could be used to detect the relative levels of phosphorylation of 26 kinases (Akt1, Akt2, Akt3, Akt pan, CREB, ERK1, ERK2, GSK-3α/β, GSK-3β, HSP27, JNK1, JNK2, JNK3, JNK pan, MKK3, MKK6, SK2, p38α, p38β, p38δ, p38γ, p53, p70 S6 Kinase, RSK1, RSK2, TOR). Briefly, antibody array membranes were incubated with protein lysates and then followed by 45
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Fig. 1. Identification of human uterine leiomyoma cells and infection with 14-3-3γ adenovirus vectors. A: Negative control that staining of α-Actin in human leiomyoma cells (×100); B: α-Actin positive staining (×100); C: Double digestion of pHBAd-MCMV-RFP vector plasmid; D: Double digestion of 14-3-3γ; E: The monoclonal PCR identification results of positive recombinant 14-3-3γclones; F: The transfection efficiency of 14-3-3γ adenovirus vectors. Each experiment was repeated at least three times.
the cells were also affected by the over-expression of 14-3-3γ. The cells of both the Wt (blank control) group and the vector (negative control) group have similar morphology of elongated shapes (Fig. 2C). However, cells in 14-3-3γ over-expression group became rounded with less elongated cytoplasmic processes (Fig. 2C). Also there are more cells in 14-3-3γ over-expression group lost attachment to the plate, assumed smaller- and rounded-shape, a typical morphology of dying and degenerating cells. To examine whether over-expression of 14-3-3γ indeed affected cell death, apoptosis were assessed by flow cytometry and TUNEL assays. As shown in Fig. 2D and E, 14-3-3γ over-expression significantly increased cell apoptosis compared to Wt and vector groups (P < 0.01), suggesting that increase in apoptosis may also play a role in the differences in the number of cells maintained between 14-3-3γ and the two control groups (Fig. 2B). These results were also confirmed in the uterine leiomyoma immortal stem cells, which got the similar conclusions.
recombinant adenovirus (pHBAd-MCMV-RFP-14-3-3γ) or control vector (pHBAd-MCMV-RFP). Insertion of 14-3-3γ gene did not affect the infection efficiency (Fig. 1F). Infection of empty vector (pHBAd-MCMVRFP) did not change 14-3-3γ protein levels compared to the cells without treatment/infection (Wt), indicating that the virus and infection, by themselves, had no effect on the expression of 14-3-3γ (Fig. 2A). In contrast, infection of cells with 14-3-3γ-containing virus (pHBAd-MCMV-RFP-14-3-3γ), at 200 MOI, significantly increased the expression of the protein (Fig. 2A). 3.2. Effects of 14-3-3γ over-expression on cell proliferation and apoptosis For the cells received control vector, they increase in numbers during the first 36 h in culture and then slowly decrease in numbers through 60 h (Fig. 2B). However, for the cells over-expressing 14-3-3γ the numbers failed to increase during the first 24 h, which decreased sharply through 60 h, so by the end of culture, only half of the cells survived compared to the control group received empty vectors. Accompanied with the differences in the cell numbers, the morphology of 46
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Fig. 2. Effects of 14-3-3γ over-expression on human uterine leiomyoma cells proliferation and apoptosis. A: The expression of 14-3-3γ protein in cells transfected with 14-3-3γ overexpression; B: The effects of 14-3-3γ over-expression on cell proliferation; C: The effects of 14-3-3γ over-expression on cell morphology; D: The effects of 14-3-3γ over-expression on cell apoptosis by flow cytometry; E: The effects of 14-3-3γ over-expression on cell apoptosis by TUNEL assays. Each experiment was repeated at least three times.
The 42 differentially expressed proteins were analyzed by PANTHER database with different functional annotations (the molecular function, biological process, protein class, cell component, signaling pathway and others) (Fig. 3A). Cross-examinations of these annotations indicate that over-expression of 14-3-3γ has major effects on cellular proteins with binding/catalytic activities and proteins affecting the structure of the cells (Fig. 3A: Molecular function). Further classification indicates proteins involved in signaling and cytoskeletal/adhesion were among the ones affected the most (Fig. 3A: protein class). Finally, more detailed signaling pathway analysis has found that the affected signaling molecules concentrated on EGF-, FGF-, insulin/IGF-,
3.3. Effects of 14-3-3γ over-expression on the global changes of protein levels To elucidate the mechanism(s) by which the cell morphology, growth and apoptosis were affected by over-expression of 14-3-3γ, the global changes in protein levels were screened by Tandem Mass Tag combined with liquid chromatography-tandem mass spectrometry. Among the total of 5846 proteins detected, 42 proteins were found affected significantly by over-expression of 14-3-3γ (Table 1). In total, 9 proteins were significantly increased by at least 1.5 fold and 33 proteins were down-regulated significantly by at least 0.7 fold. 47
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pathway after 14-3-3γ over-expression, mTOR, TSC2 and Foxo1 proteins were examined because of their close relations to the AKT signaling pathway. As shown in Fig. 4B, 14-3-3γ over-expression significantly reduced the phosphorylations of AKT (p-AKT) and Foxo1 (pFoxo1), but not of mTOR (p-mTOR) or of TSC2 (p-TSC2). Also, 14-3-3γ over-expression did not affect the total protein levels of any of these 4 targets. Again, these results confirmed the above observations that 143-3γ affects the signaling pathways mostly by phosphorylation, not by synthesis/degradation of the protein themselves. Nuclear transportation of transcriptional factors is one of the critical steps for most of the signaling transduction processes. To examine whether 14-3-3γ over-expression could affect the cellular redistribution of the effecting factor Foxo1, nuclear and cytoplasm were isolated and the amounts of Foxo1 and p-Foxo1 were analyzed by Western blots in the two fractions (Fig. 5A). Relatively, the expression of both p-Foxo1 and Foxo1 were higher in nuclear than in cytoplasm for the vector and 14-3-3γ over-expression groups (P < 0.05). When the proteins were expressed as nuclear/cytoplasmic ratios, p-Foxo1 was significant reduced by 14-3-3γ over-expression (P < 0.01) compared to the vector group, while there was no significant difference of Foxo1 between the two groups (Fig. 5B, C). These results indicated that in addition to affect the overall phosphorylation of Foxo1, 14-3-3γ over-expression may also affect the distributions of p-Foxo1. It can specifically reduce the nuclear localization of pFoxo1. To further explore the possible changes in the upstream proteins of ERK1/2 pathway, phosphorylations of MEK1/2 were also examined (Fig. 6A). As shown in Fig. 6A, p-MEK1/2 and p-ERK1/2 were both down-regulated by 14-3-3γ over-expression (P < 0.01), while the total protein levels of MEK1/2 and ERK1/2 were not affected significantly, suggesting that loss of p-MEK1/2 may be the reason that p-ERK1/2 were reduced. In addition to the signaling molecules, two of the most important cell structure proteins, vimentin and desmin were also analyzed. Overexpression of 14-3-3γ did not affect the total protein levels of both vimentin and desmin, but significantly reduced the phosphorylated form of vimentin (p-Vimentin), but not desmin (Fig. 6B).
Table 1 The obvious changed protein when 14-3-3γ over-expression in uterine leiomyoma. Accession no.
Protein
Fold
Expression
P61981 P20591 P05362 P31947-2 O15078 P48307-2 P04179-4 P43490 A0A087WW59 F8WCR8 P49448 O75157-2 Q15582 Q04837 Q8IVF2 H3BM79 P17661 P31749 H7BY57 Q04917 Q8IZV5 O43524-2 Q12778 P09486 Q96B36-2 P29373 Q08AE8-2 Q15149-4 P31751 O14828-2 A0A087WW43 F5GXS0 B4DLN1 Q9BTT0-3 P08670 P27216 A0A0A0MT32 P01023 P02452 H0YN26 P04731 E9PND2
14-3-3 protein γ MX1 ICAM1 14-3-3 protein sigma CEP290 TFPI2 SOD2 NAMPT C11orf96 NCKIPSD GLUD2 TSC22D2 TGFBI SSBP1 AHNAK2 AKTIP Desmin AKT1 NFASC 14-3-3 protein eta RDH10 Foxo3 Foxo1 SPARC AKT1S1 CRABP2 SPIRE1 PLEC AKT2 SCAMP3 ITIH3 C4B Uncharacterized protein ANP32E Vimentin ANXA13 LIPA A2M COL1A1 ANP32A MT1A CSRP1
4.238 1.930 1.859 1.774 1.616 1.576 1.573 1.517 1.511 0.700 0.698 0.698 0.697 0.693 0.689 0.689 0.689 0.678 0.676 0.669 0.669 0.669 0.667 0.665 0.664 0.664 0.663 0.660 0.654 0.649 0.644 0.644 0.639 0.633 0.632 0.617 0.613 0.608 0.597 0.525 0.515 0.311
↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓
4. Discussion Despite the high prevalence of uterine leiomyomas in human population, which affects millions of woman around the world, their real cause remains unknown [1]. In the previous study, we have found that 14-3-3γ protein was down-regulated significantly in uterine leiomyoma compared to the surrounding normal myometrium [3]. We hypothesize that loss in 14-3-3γ protein may play a causal role in the growth/progression of uterine leiomyoma. To test this hypothesis, we have restored the loss of 14-3-3γ protein in uterine leiomyoma cells by overexpressing the protein through an adenoviral vector, which was constructed by our lab. Infection of uterine leiomyoma cells with the vector successfully resulted in significant increases in 14-3-3γ protein level in the cells. The transgenic approach largely normalized the level of 14-3-3γ in uterine leiomyoma cells to that of normal myometrium. Interestingly, restoration of 14-3-3γ significantly inhibited cell proliferation and promoted cell apoptosis. These results support our hypothesis that loss of 14-3-3γ may indeed play a causal role in the growth/progression of uterine leiomyoma. To elucidate the molecular mechanism involved, we have screened the changes in the protein levels, at globe scale, in the overexpressing cells and the phosphorylations of key members of a few well-known 143-3γ related signaling pathways. With the latest proteomics technology, 42 differentially expressed proteins were identified from a total of 5846 proteins examined. Bioinformatical analysis of the 42 differentially expressed proteins by PANTHER database and GeneMANIA, have revealed that the differentially regulated proteins are involved in variety biological processes, mostly in signaling transductions and structure
PI3- and chemokine/cytokine-pathways (Fig. 3A: signal pathway). To determine the interrelationship among these 42 genes, GeneMANIA network analysis was applied, the results of which are displayed in Fig. 3B. The derived network consisted of 22 of the 42 core genes and 14 additional genes that were pulled in by GeneMANIA. These results are consistent with the observations that 14-3-3γ functions as an adopt protein that is capable of modifying a broader range of signaling transductions.
3.4. Effects of 14-3-3γ over-expression on the signaling transductions of MAPK, AKT and Foxo1 To further examine how 14-3-3γ over-expression affect the key signaling pathways, which are potentially important to cell proliferation and apoptosis, phosphorylations of the key members of MAPK, AKT and Foxo1 were analyzed by phospho-MAPK pathway array and Western blots. As shown in Fig. 4A, over-expression of 14-3-3γ resulted in reductions in the phosphorylations of multiple members of the signaling molecules, including p-AKT1, p-AKT2, p-AKT pan, p-ERK1, p-GSK-3α and p-GSK-3β, while the protein themselves were not affected significantly, suggesting that 14-3-3γ affects the signaling pathways mostly by phosphorylation, not by synthesis/degradation of the protein themselves. These results were mostly confirmed by Western blots. To further explore the changes of the downstream proteins of AKT 48
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Fig. 3. The bioinformatics analysis of differentially expressed proteins after 14-3-3γ over-expression by PANTHER and GeneMANIA. A: The bioinformatics analysis of differentially expressed proteins after 14-3-3γ over-expression by PANTHER database with different functional annotations (the molecular function, biological process, protein class, cell component, signaling pathway and others); B: The interrelationship of differentially expressed proteins after 14-3-3γ over-expression by GeneMANIA network analysis.
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Fig. 4. Effects of 14-3-3γ over-expression on the signaling transductions of MAPK, AKT and Foxo1. A: Effects of 14-3-3γ over-expression on phospho-MAPK related proteins; B: Effects of 14-3-3γ over-expression on the expression of mTOR, TSC2, Foxo1 and AKT. Each experiment was repeated at least three times.
phosphorylation of ERK1/2. Such effect may result from the actions on the up-stream of the signaling pathway since accompanied by decrease in p-ERK1/2, p-MEK1/2 was also reduced by over-expression of 14-33γ. At present, however, it is not clear how 14-3-3γ inhibits MEK1/2 phosphorylations. Overall, the results suggest that inhibition of MEK1/ 2-ERK1/2 signaling could be one of the mechanisms by which 14-3-3γ inhibits uterine leiomyoma cell proliferation and induces their apoptosis. This conclusion is also supported by findings in breast epithelial cells, where 14-3-3γ could reduce the cellular damage caused by lipopolysaccharide through inhibiting the ERK1/2 signaling [25]. In addition to MAPK, AKT is another signaling pathway that plays a central role in regulating cell survival, proliferation, apoptosis and migration. Evidences suggest that activation of this signaling may also play a role in the growth/progression of uterine leiomyoma. For example, Xu et al. [26] found that the expression of p-AKT in uterine leiomyoma was significantly increased compared to that in surrounding normal myometrium tissues, and that blocking the signaling in uterine leiomyoma cells significantly increased cell apoptosis. Similarly, inhibition of PI3K (an up-stream kinase that phosphorylates AKT) can increase uterine leiomyoma cell apoptosis and promote cell cycle arrested [27]. Although the involvement of AKT signaling in the growth/ progression of uterine leiomyoma seems clear, the mechanisms/factors that increase AKT and/or its phosphorylation is still unknown. In our
regulations. However, these changes in the quantity of proteins, by themselves, cannot explain the overall changes (proliferation, apoptosis and morphology) observed in the cells. To elucidate more detailed mechanisms involved, the phosphorylation of the two major signaling pathways, MAPK and AKT, have been examined. These pathways were selected for three reasons: 1) They are closely related to cell proliferation and apoptosis; 2) They are among the most common pathways modified by 14-3-3γ; 3) They are frequently modified by treatments/manipulations that affected uterine leiomyoma cell proliferation and/or apoptosis. ERK1/2 is one of the best studied MAPK signaling pathway members (Ras/Raf/MEK/ERK pathway). It is well known that this pathway plays significant roles in the regulation of cell proliferation and apoptosis [21,22]. Chegini et al. reported that the expression of ERK1/2 protein was increased in uterine leiomyoma, compared to the surrounding normal myometrium [23]. Such increase most likely plays a causal role in uterine leiomyoma growth since inhibition of the signaling (MEK1/2-ERK1/2) significantly reduced the proliferation of uterine leiomyoma cells [10,23,24]. However, the mechanism that is responsible for the up-regulation of ERK1/2 or its phosphorylation is still unknown. In the present study, we have found that the loss of 14-33γ may play a role in the increase of p-ERK1/2 in uterine leiomyoma cells since increase/restoration of 14-3-3γ significantly reduced the
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Fig. 5. Effects of 14-3-3γ over-expression on the nuclear and cytoplasmic expression of Foxo1 protein. A: Effects of 14-3-3γ over-expression on the nuclear and cytoplasmic expression of p-Foxo1 and Foxo1 proteins by Western blot; B: Effects of 14-3-3γ over-expression on the nuclear and cytoplasmic expression of p-Foxo1 and Foxo1 proteins; C: Effects of 14-3-3γ overexpression on the nuclear/cytoplasmic expression of p-Foxo1 and Foxo1 proteins. Each experiment was repeated at least three times.
has been frequently observed in other systems. For example, it is well known that 14-3-3 protein can carry its physiological function and keep the dynamic balance of cell apoptosis through affecting the phosphorylation of AKT at threonine 308 site [28]. In human breast cancer cells, 14-3-3σ up-regulation could inhibit cell proliferation and tumor
present study, we have found that loss of 14-3-3γ may play a causal role in the activation of AKT signaling since increase/restoration of 14-3-3γ in uterine leiomyoma cells significantly reduced the phosphorylation of AKT and its down-stream effectors. The regulatory relationship between 14-3-3γ and AKT is not special to uterine leiomyoma cells since it
Fig. 6. Effects of 14-3-3γ over-expression on the expression of selected potential function proteins. A: Effects of 14-3-3γ over-expression on the expression of p-MEK1/2, MEK1/2, p-ERK1/ 2 and ERK1/2 proteins by Western blot; B: Effects of 14-3-3γ over-expression on the TGFBI, p-Vimentin, Vimentin and Desmin proteins by Western blot. Each experiment was repeated at least three times.
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progression of human uterine leiomyoma. Compared to previous studies, there are some new discoveries in this manuscript. Increase/restoration of 14-3-3γ in uterine leiomyomal cells reduced cell proliferation and increased cell apoptosis. AKT-Foxo, MEK1/2-ERK1/2 and vimentin signaling may be involved, while TSC2, mTOR, TGFBI and Desmin are unlikely to play any significant role in the process. The mechanism that is responsible for the loss of 14-3-3γ in human uterine leiomyoma, however, is still unknown and deserves for further study.
progression via inhibiting AKT activation [29]. In the breast epithelial cells, 14-3-3γ up-regulation can reduce the cell damage induced by lipopolysaccharide through the activation of AKT pathway [25]. Foxo, the downstream effecting molecule of AKT pathway, often functions as a tumor suppressor. Its function can be negatively regulated by Akt-mediated phosphorylation, since p-Foxo is often transported from nucleus into cytoplasm, where it is bound with 14-3-3 protein, and prevented from binding to target gene transcriptions [30]. Kovács et al. [31] have found that the expressions of p-AKT and pFoxo1 were both increased in uterine leiomyoma, and that the p-Foxo1 in the nucleus was significantly higher in leiomyoma than in normal myometrium. Foxo is a 14-3-3 protein ligand, through which 14-3-3 protein can regulate nucleus/cytoplasm distribution of Foxo [6]. Also, 14-3-3 protein can affect cell proliferation and apoptosis by preventing the Foxo from degradation or dephosphorylation [12]. In this study, we have found that both p-AKT and p-Foxo was significantly inhibited by over-expression of 14-3-3γ, and p-Foxo nucleus/cytoplasm expression ratio was also affected (decreased). We found that 14-3-3γ could not only decrease the overall phosphorylation of Foxo1, but also reduce the nuclear localization of p-Foxo1. This mechanism may be that over-expression of 14-3-3γ firstly decreased the total p-AKT, then total p-Foxo was decreased; 14-3-3γ also regulated the p-Foxo transportation from nucleus into cytoplasm. These changes relatively increased the level of Foxo, which functions as a tumor suppressor. These observations suggest strongly that p-AKT/p-Foxo down-regulation could be one of the likely reasons that over-expression of 14-3-3γ resulted in increasing cell apoptosis and decreasing cell proliferation in uterine leiomyoma. Vimentin is a member of the intermediate filament family, which in addition to maintain the integrity of the cell, can involve in cell migration, adhesion, signal transduction, and cell apoptosis [32]. Vimentin is expressed by a variety of tumor cells and may be served as a marker for assessing the degrees of cancer malignancy [33]. Vimentin can regulate 14-3-3 activities by binding to the protein when phosphorylated, which inhibits the ability of 14-3-3 binding to other targets [34]. Vimentin, AKT and 14-3-3 can form a complex to participate in tumor cell autophagy and apoptosis. Also, vimentin can be regulated by the ERK1/2 pathway [35]. We have found that the expression of vimentin was also increased in uterine leiomyoma compared to the surrounding normal myometrium. In the present study, the decrease in pVimentin protein by over-expression 14-3-3γ suggests that vimentin could be another player in the promotions of growth/progression of uterine leiomyoma cells by loss of 14-3-3γ. In addition to Foxo1 and vimentin, this study also examined other potential targets and/or modifying members of AKT and/or ERK1/2 signaling. These include: TSC2, a tumor suppressor, locating at the downstream of the AKT pathway [36]; mTOR, another important downstream target of AKT [37,38], which also was up-regulated in uterine leiomyomas [39]; TGFBI, an important regulator in tumorigenesis of a broader range of systems [40], which was also up-regulated in uterine leiomyoma [3]; and Desmin, a cytoskeletal protein, often being used as one of the markers of cell proliferation and differentiation in early stage [41], which was also up-regulated in uterine leiomyoma [3]. Although most of these potential target proteins were increased in uterine leiomyoma, over-expression of 14-3-3γ did not significantly affect the levels of the total proteins or phosphorylations, suggesting that they are unlikely the targets and/or mediators in 14-3-3γ induced down-regulation of cell apoptosis and up-regulation of cell divisions. Besides, in our previous study [42], we have found that 14-3-3γ promoter was methylated more in leiomyomas than in matched myometrium, and 14-3-3γ was negatively correlated with ER and PR proteins, whose expressions were higher in leiomyomas. These results may suggest that the hypermethylation of the 14-3-3γ promoter may account, at least partially, for the down-regulation of 14-3-3γ in leiomyomas. In summary, 14-3-3γ expression is reduced in human uterine leiomyoma, which may be a causal factor in the development and/or
Funding This research was supported by a grant from the National Natural Science Foundation of China (No. 81171926) and Zhejiang Provincial Natural Science Foundation of China/Zhejiang Provincial Basic Public Welfare Research Project under Grant No. LQ18H160026. Acknowledgments We thank Professor Haolin Chen at Department of Biochemistry, Johns Hopkins University, Bloomberg School of Public Health (USA) for editing the manuscript. Conflict of interest The authors declare no conflict of interest. References [1] E.E. Wallach, N.F. Vlahos, Uterine myomas: an overview of development, clinical features, and management, Obstet. Gynecol. 104 (2004) 393–406. [2] S. Sankaran, I.T. Manyonda, Medical management of fibroids, Best Pract. Res. Clin. Obstet. Gynaecol. 22 (2008) 655–676. [3] J. Lv, X. Zhu, K. Dong, Y. Lin, Y. Hu, C. Zhu, Reduced expression of 14-3-3 gamma in uterine leiomyoma as identified by proteomics, Fertil. Steril. 90 (2008) 1892–1898. [4] A. Aitken, 14-3-3 proteins: a historic overview, Semin. Cancer Biol. 16 (2006) 162–172. [5] S. Wang, X. Li, Z.G. Li, J. Lu, W.Y. Fang, Y.Q. Ding, et al., Gene expression profile changes and possible molecular subtypes in differentiated-type nonkeratinizing nasopharyngeal carcinoma, Int. J. Cancer 128 (2011) 753–762. [6] D.K. Morrison, The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development, Trends Cell Biol. 19 (2009) 16–23. [7] Y. Song, Z. Yang, Z. Ke, Y. Yao, X. Hu, Y. Sun, et al., Expression of 14-3-3γ in patients with breast cancer: correlation with clinicopathological features and prognosis, Cancer Epidemiol. 36 (2012) 533–536. [8] P. Raungrut, A. Wongkotsila, K. Lirdprapamongkol, J. Svasti, S.L. Geater, M. Phukaoloun, et al., Prognostic significance of 14-3-3γ overexpression in advanced non-small cell lung cancer, Asian Pac. J. Cancer Prev. 15 (2014) 3513–3518. [9] S. Liang, G. Shen, Q. Liu, Y. Xu, L. Zhou, S. Xiao, et al., Isoform-specific expression and characterization of 14-3-3 proteins in human glioma tissues discovered by stable isotope labeling with amino acids in cell culture-based proteomic analysis, Proteomics Clin. Appl. 3 (2009) 743–753. [10] E.N. Nierth-Simpson, M.M. Martin, T.C. Chiang, L.I. Melnik, L.V. Rhodes, S.E. Muir, et al., Human uterine smooth muscle and leiomyoma cells differ in their rapid 17 beta-estradiol signaling: implications for proliferation, Endocrinology 150 (2009) 2436–2445. [11] A.V. Hoekstra, E.C. Sefton, E. Berry, Z. Lu, J. Hardt, E. Marsh, et al., Progestins activate the AKT pathway in leiomyoma cells and promote survival, J. Clin. Endocrinol. Metab. 94 (2009) 1768–1774. [12] J. Du, W. Liao, Y. Wang, C. Han, Y. Zhang, Inhibitory effect of 14-3-3 proteins on serum-induced proliferation of cardiac fibroblasts, Eur. J. Cell Biol. 84 (2005) 843–852. [13] L. Ting, R. Rad, S.P. Gygi, W. Haas, MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics, Nat. Methods 8 (2011) 937–940. [14] J. Sinclair, J.F. Timms, Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS, Methods 54 (2011) 361–369. [15] R.M. Sturm, C.B. Lietz, L. Li, Improved isobaric tandem mass tag quantification by ion mobility-mass spectrometry, Rapid Commun. Mass Spectrom. 28 (2014) 1051–1060. [16] D.J. Clark, W.E. Fondrie, Z. Liao, P.I. Hanson, A. Fulton, L. Mao, et al., Redefining the breast cancer exosome proteome by tandem mass tag quantitative proteomics and multivariate cluster analysis, Anal. Chem. 87 (2015) 10462–10469. [17] H.J. Lee, H.J. Cha, J.S. Lim, S.H. Lee, S.Y. Song, H. Kim, et al., Abundance-ratiobased semiquantitative analysis of site-specific N-linked glycopeptides present in the plasma of hepatocellular carcinoma patients, J. Proteome Res. 13 (2014) 2328–2338. [18] E. Maes, D. Valkenborg, I. Mertens, V. Broeckx, G. Baggerman, X. Sagaert, et al.,
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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
Overexpression of the 14-3-3γ protein in uterine leiomyoma cells results in growth retardation and increased apoptosis ⁎
Qi Shena, Xiaoli Hua, Lulu Zhoua, Shuangwei Zoua, Lu-Zhe Sunb, , Xueqiong Zhua, a b
T
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Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China Department of Cell Systems & Anatomy, University of Texas Health Science Center at San Antonio, TX, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Uterine leiomyoma 14-3-3γ Proliferation Apoptosis Bioinformatics Signaling pathway
Protein 14-3-3γ was significantly reduced in human uterine leiomyoma compared to the adjacent normal myometrium tissue. To investigate the possible link between the reduced 14-3-3γ expression and uterine leiomyoma growth, we have overexpressed 14-3-3γ protein in uterine leiomyomal cells and its effects on cell proliferation and apoptosis were analyzed. Over-expression of 14-3-3γ was achieved by transducing into two types of uterine leiomyoma cells (primary culture cells and immortal stem cells) with a 14-3-3γ expressing adenovirus vector. Differentially expressed proteins were screened by the proteomics tool (TMT-LCTMS), followed by PANTHER database analysis to single out specifically modified signaling pathway proteins, which were confirmed by Phospho-MAPK Antibody Array and Western blots analysis. The results showed that increase in 143-3γ expression in both two types of human uterine leiomyoma cells inhibited cell proliferation and induced apoptosis. Proteomic screening has found 42 proteins, among 5846, that were significantly affected. PANTHER database and GeneMANIA analysis of the differentially expressed proteins have found that proteins involved in apoptosis signaling and cytoskeletal/adhesion were among the ones affected the most. Further analysis of the key signaling pathways have found that over-expression of 14-3-3γ resulted in reductions in the phosphorylations of multiple signaling molecules, including AKT, pan, ERK1/2, GSK-3 α/β, MEK1/2, Foxo1 and Vimentin. In conclusion, the loss of 14-3-3γ may have causal effects on the growth of uterine leiomyoma, which may function through modifying multiple signaling pathways, including AKT-Foxo and/or MEK1/2-ERK1/2.
1. Introduction Uterine leiomyomas are among the most common tumors in women, affecting 20% to 50% of reproductive women, which can cause menorrhagia, abnormal uterine bleeding, pelvic pain, infertility and miscarriage [1]. Except invasive surgical procedure, there is no simple, safe and effective way to treat the disease currently. Uterine leiomyomas are generally considered as a hormonal dependent tumor, closely related to estrogen and progesterone, but the mechanism of tumorigenesis is still unclear [2]. In our previous study, we have found that 14-3-3γ exhibited a marked down-regulation in leiomyoma tissues compared with the adjacent normal myometrium, via proteomic and reverse transcription polymerase chain reaction (RT-PCR) techniques [3]. In this study, we have tested the hypothesis that the loss of 14-3-3γ protein may play a causal role in the origin or growth of leiomyoma.
14-3-3γ is a member of 14-3-3 proteins family (7 isoforms: β, γ, ε, η, σ, τ/θ and ξ), a highly conserved phosphoserine/threonine-binding proteins group that regulates diverse cellular processes, including cell cycle progression, transcriptional regulation, apoptosis, and cell proliferation [4,5]. 14-3-3 proteins have been found to play roles in human tumorigenesis, with different subtypes expressed in different cancer tissues and possible different regulatory mechanisms involved [6]. At present, 14-3-3γ has been reported in breast cancer, lung cancer and glioma [7–9]. However, the role of 14-3-3γ in gynecological tumors has not been reported. Mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signal transduction pathways are capable of regulating the proliferation and apoptosis of uterine leiomyoma cells, which are considered to be the main signal pathways that are responsible for the development of uterine leiomyomas [10,11]. In
Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; MAPK, mitogen-activated protein kinases; PI3K/AKT, and phosphatidylinositol 3-kinase/protein kinase B; ERK1/2, extracellular regulated protein kinases 1/2; TMT, tandem mass tag; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; CCK-8, cell counting kit-8; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel; LSD, least significance difference ⁎ Corresponding authors at: No. 109 Xueyuan Xi Road, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China. E-mail addresses: [email protected] (L.-Z. Sun), [email protected] (X. Zhu). https://doi.org/10.1016/j.cellsig.2018.01.025 Received 17 December 2017; Received in revised form 22 January 2018; Accepted 25 January 2018 0898-6568/ © 2018 Published by Elsevier Inc.
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culture medium above, under 2.5% O2 and 5% CO2 at 37 °C. Culture medium was changed every other day. Cells were split as 1:2 every 4 to 6 days. Cells from third passages to the fifth were used for the experiments.
uterine leiomyoma cells, for example, estrogen is capable of increasing cell proliferation via protein kinase C, which further activates the extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway. Interestingly, in normal myometrium cells, estrogen is also capable of activating protein kinase C, but instead of stimulating MAPK/ERK1/2 signaling, the kinase inactivates MAPK/ERK1/2 pathway, which resulted in low level of cell proliferation activities. The difference in the signaling transductions of the two cells/systems is still unclear. Progesterone induces PI3K/AKT pathway to activate the downstream genes (GSK3, TSC2, Foxo, and others), which can inhibit the leiomyoma cells apoptosis and also promote cell proliferation [11]. 14-3-3γ is a binding partner of both downstream members of MAPK (such as JNK, p38 and ERK1/2) and PI3K/AKT (such as Foxo, BAD and BAX), playing an important role in cell proliferation and apoptosis via preventing the members from dephosphorylation, which further controls the proportion of cytoplasmic and nuclear protein distributions [12]. In this study, we tested a hypothesis that loss of 14-3-3γ by uterine leiomyoma cells plays a causal role in the development of leiomyoma by modifying MAPK and/or PI3K/AKT signaling pathways. Tandem mass tag (TMT) is a new proteomics technology that can quantify the absolute amount of proteins by specifically labeling the peptides and proteins [13,14]. Compared to the traditional bidirectional gel electrophoresis proteomics, TMT has clear advantages in detection range, reproducibility, high flux, accuracy and high resolution [15]. Since its invention, TMT has been used widely in cancer researches, such as breast-, liver-, and colorectal-cancers and others [16–18]. In this study, TMT was used to identify differently expressed proteins in cells after 14-3-3γ up-regulated, phospho-MAPK antibody array and Western blots were used for further conformation. Overall, our study tried to explore the role of 14-3-3γ in leiomyoma cell proliferation and apoptosis, and related signal pathways, which will help us better learn the biological effects of 14-3-3γ, and also provide experimental basis for clinical treatment on uterine leiomyoma patients.
2.3. Staining of α-smooth muscle actin by immunocytochemistry Uterine leiomyoma cells were identified by the expression of αsmooth muscle actin. Cells were fixed with 4% paraformaldehyde, washed and then permeabilized in PBS containing 0.2% Triton X-100 for 15 min. Cells were then incubated in a serum-free blocking solution for 15 min at room temperature, incubated with mouse monoclonal anti-α-smooth muscle actin antibody (1:100 dilution; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) overnight at 4 °C. After extensive washing with PBS, cells were incubated with biotinylated goat anti-mouse IgG as secondary antibody. After incubation the bound antibodies were visualized using 3,3′-diaminobenzidine. Finally, nuclei were stained with hematoxylin. Cells incubated with PBS, instead of primary antibody, was included as negative control. 2.4. Construction and characterization of recombinant plasmid vectors Human 14-3-3γ (Genebank: NM_012479.3) gene was synthesized by GenScript company (Shanghai, China). The vector plasmid (pHBAdMCMV-RFP, Hanbio, Shanghai, China) was digested by the BamHI and EcoRI restrictive enzymes, and purified using agarose gel electrophoresis. Then 14-3-3γ gene was subcloned into the pHBAd-MCMV-RFP. The positive recombinant clone, named pHBAd-MCMV-RFP-14-3-3γ, was then transfected and amplified into DH5α by hot shock method and screened by Amp + resistance. The positive clones were finally identified and characterized by PCR and sequencing. 2.5. Virus preparation, purification, and titer determination The pHBAd-MCMV-RFP-14-3-3γ and the adenovirus helper plasmid pHBAd-BHG (Hanbio, Shanghai, China) were co-transformed into HEK293 cells with lipofectamine 2000 Kit according to the manufacturer's instructions. Six hours after transformation, the medium was replaced with new DMEM, and cytopathic effects were observed every 2 days. The viral particles in the HEK293 cells were prepared by lysis of the cells with three consecutive freeze–thawing cycles in ice-ethanol, then by passing through 0.4 μM syringe filter to remove inadvertent materials. The viral titer (pHBAd-MCMV-RFP-14-3-3γ) was detected by the TCID50 standard method. Finally, the recombinant adenovirus was stored at −80 °C until use. pHBAd-MCMV-RFP-14-3-3γ virus was used to over-express 14-3-3γ proteins, while pHBAd-MCMV-RFP virus was used as a negative control.
2. Materials and methods 2.1. Primary tissue collection and stem cell Uterine leiomyoma tissues were obtained from 40 patients with ages ranged 43 ± 6 years, between October 2013 and July 2015. The tissues were collected during the procedures of uterine myomectomy, subtotal or total hysterectomy. Patients with following characters were excluded: administrations with medicines or hormones within 3 months prior surgery, or with other complications, such as infections, chronic diseases (such as diabetes and hypertension), uterine malignancy and/ or adenomyosis (on the basis of tissue pathology). The study protocol was approved by the Research Ethical Committee of the Second Affiliated Hospital of Wenzhou Medical University and met the standards of the Declaration of Helsinki. Written consent was obtained from every patient before the procedures. Human uterine leiomyoma stem cells were from Dr. Ayman AlHendy Lab, we have immortalized them with hTERT lentivirus.
2.6. Cell counting kit-8 (CCK-8) assay To detect the cell proliferation, 1 × 104 cells/well were seeded into 96-well plates and incubated at 37 °C with 5% CO2. After 24 h of incubation, the cells were treated with 14-3-3γ over-expression virus or control virus. By end of treatments, 10 μl of CCK-8 (Dojindo, Kumamoto, Japan) was added to each well and incubated for an additional 2 h. The reaction product was quantified by spectrophotometry at 450 nm wavelength, and the percentage of viability or number of cells were calculated by formula: (treated cells absorbent / non treated cells absorbent) × 100.
2.2. Primary human cell and stem cell culture Fresh tissues were washed with cold phosphate-buffered saline (PBS), then cut into small pieces (1 mm3) and digested with 0.2% (v/v) collagenase II (Invitrogen, Carlsbad, CA, USA) in Dulbecco's modified Eagle's medium (DMEM) for 4 h in a 37 °C with shaking. The dissociated cells were centrifuged at 400 ×g for 5 min. The resultant cell deposit was suspended with complete culture medium (DMEM, 10% fetal bovine serum, 100 IU/ml of penicillin G and 100 μg/ml streptomycin) and centrifuged at 400 ×g for 5 min. The resultant cells were cultured at a density of 2 × 105 cells/ml under 5% CO2 at 37 °C in the complete culture medium. Stem cells were cultured with the same complete
2.7. Flow cytometric assay of apoptosis with APC/7-AAD staining Cell apoptosis was assayed by flow cytometry after APC/7-AAD staining. APC was used to detect early apoptosis in cellular membrane, 7-AAD was used to detect late apoptosis in cell nucleus. The assay was performed according to the manufacturer's guidelines. Briefly, after 44
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biotinylated antibodies. Finally the membranes were incubated with streptavidin horseradish peroxidase-conjugated antibody. Immunoreactivity was visualized using a chemiluminescent substrate. Densitometric analysis was performed using GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, USA).
washed twice with cold PBS and then re-suspended in 1× binding buffer, cells (105 cells in 100 μl of the solution) were mixed with 5 μl of APC and 5 μl 7-AAD. After gently vortexed, the cells were incubated for 15 min at room temperature (25 °C) in the dark and then were added 200 μl of 1× binding buffer to each tube, and then analyzed by flow cytometry within 1 h.
2.11. TMT combined with liquid chromatography-tandem mass spectrometry
2.8. Apoptosis assay by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining
Cells were lysed in Radio-Immunoprecipitation Assay lysis buffer and sonicated. The protein lysates were denatured, reduced, and alkylated as recommended by the TMT Mass Tagging Kit (Thermo Fisher Scientific, Waltham, MA, USA) protocol. Trypsin was added at a protein/enzyme ratio of 30:1 by mass and the digestion was performed at 37 °C overnight. Peptides were labeled with TMT reagents according to the manufacturer's protocol in duplicate and equal amounts of labeled peptides were combined to obtain one sample, which was separated into 9 fractions by strong cation exchange chromatography using TopTip columns (PolyLC). Elution was performed with increasing concentrations of KCl (from 0 to 0.5 M). Eluates were dried using a SpeedVac and then desalted using C18/hypercarb PolyLC. Samples from each fraction were run in triplicate on an Orbitrap Pro mass spectrometer that was coupled to a nanoLC (Thermo Fisher Scientific), and the spectra obtained were analyzed with Proteome Discoverer 1.3 software against the Human International Protein Index (IPI) database. Peptides were identified with a false discovery rate of < 1%.
For the analysis of cell death, apoptotic cells were labeled with TUNEL staining (Roche, Indianapolis, IN, USA). TUNEL was used to detect apoptosis in cell nucleus. The procedure was done according to the manufacturer's instruction. The apoptotic cells were identified by nuclear staining of dark brown colors, which was quantified by counting 100 cells from six random microscopic fields in each group, and the values were presented as the percentage of positive cells in the total number of cells. 2.9. Cell lysate preparation and Western blot analysis Total proteins were prepared by whole-cell lysis with the buffer (60 mM Tris-HCl, pH 6.8, 5% glycerol, 2% SDS) on ice. Cytoplasmic and nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, IL, USA) according to the manufacture's protocol. The protein from each experimental group was quantified by bicinchoninic acid method (Beyotime, Jiangsu, China). Cellular proteins (30 μg) were solubilized in sample buffer (4% SDS, 30 mm dithiothreitol, 0.25 m sucrose, 0.01 m EDTA-Na2 and 0.075% bromophenol blue), and heated at 100 °C for 5 min to denature proteins. The proteins were separated using electrophoresis on 12% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) and then electro-transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 2 h at room temperature in 0.05 M Tris-buffered saline with 0.5% triton X-100 (TBS-T, pH 7.4) containing 5% skim milk and then incubated in TBS-T overnight at 4 °C with one of the appropriate primary antibodies at 1:1000 dilutions, unless specified otherwise. The primary antibodies used were 14-3-3γ (Santa Cruz Biotechnology, Santa Cruz, CA, USA); AKT (CST, Boston, USA); p-AKT (CST, USA); Foxo1 (CST, USA); p-Foxo1 (CST, USA); ERK1/2 (CST, USA); p-ERK1/2 (CST, USA); MEK1/2 (CST, USA); p-MEK1/2 (CST, USA); Vimentin (Abways, Shanghai, China); p-Vimentin (Abways, China); TSC2 (CST, USA); p-TSC2 (CST, USA); mTOR (Abways, China); p-mTOR (Abways, China); TGFBI (Abways, China); Desmin (Abways, China); Lamin B (Bioworld, St. Louis Park, MN, USA); β-Actin (1: 2000 dilution; Beyotime, China); and α-Tubulin (1:2000 dilution; Beyotime, China). After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 for anti-rabbit-IgG or anti-mouse-IgG) for 1 h at room temperature. Blots then were developed by an enhanced chemiluminescence. The protein bands were relatively quantified by densitometry scanning using the AlphaEaseFC 4.0 software (Alpha Innotech Corp, San Leandro, CA, USA), then normalized by corresponding levels of α-Tubulin or β-Actin respectively. Each experiment was repeated at least three times.
2.12. Bioinformatic analysis Biological processes, molecular functions and the pathways of the differentially expressed proteins involved were analyzed by PANTHER (Protein annotation through evolutionary relationship classification system; http://www.pantherdb.org/) consortium databases [19]. The combination of gene set enrichment analysis and network analysis were analyzed by GeneMANIA [20]. 2.13. Statistical methods All statistical analyses were performed with SPSS17.0 software. Data was presented as the mean ± standard deviation. Difference between two groups was analyzed by Student's t-test. Differences among multiple groups were analyzed by one-way ANOVA. If variances were homogeneous, then least significance difference (LSD) method was used to compare between two groups. If variances were nonhomogeneous, Dunnett's T3 method was applied to compare between two groups. A 2tailed P value < 0.05 was considered statistically significant. 3. Results 3.1. Culture of human uterine leiomyoma cells and infection with 14-3-3γ adenovirus vectors Human uterine leiomyoma cells were isolated from uterine leiomyoma tissues of patients aged 43 ± 6 years-old by collagenase digestion and step-wise centrifugations. A uniformed population with smooth muscle cell characteristics was achieved after 2–3 passages. To confirm the purity of the cells, immunocytochemistry staining of the cells with α-actin antibody was performed. As shown in Fig. 1(A, B), the positive staining of this smooth muscle-specific actin indicated that almost 100% of cells derived from uterine leiomyoma tissues retained their smooth muscle characteristics. To generate a virus vector expressing human 14-3-3γ gene, pHBAdMCMV-RFP plasmids were digested by the BamHI and EcoRI, then 14-33γ was subcloned into the vector. The positive recombinant clones were selected, confirmed by PCR and sequencing (Fig. 1C, D, E). The uterine leiomyoma cells at the passage 3 were infected with 14-3-3γ
2.10. Human phospho-MAPK antibody array MAPK protein phosphorylation was screened with 300 μg of cell extracts using Human Phospho-MAPK Array Kit according to the manufacturer's instructions (Proteome Profiler; R&D Systems, Minneapolis, MN, USA). Human Phospho-MAPK Array could be used to detect the relative levels of phosphorylation of 26 kinases (Akt1, Akt2, Akt3, Akt pan, CREB, ERK1, ERK2, GSK-3α/β, GSK-3β, HSP27, JNK1, JNK2, JNK3, JNK pan, MKK3, MKK6, SK2, p38α, p38β, p38δ, p38γ, p53, p70 S6 Kinase, RSK1, RSK2, TOR). Briefly, antibody array membranes were incubated with protein lysates and then followed by 45
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Fig. 1. Identification of human uterine leiomyoma cells and infection with 14-3-3γ adenovirus vectors. A: Negative control that staining of α-Actin in human leiomyoma cells (×100); B: α-Actin positive staining (×100); C: Double digestion of pHBAd-MCMV-RFP vector plasmid; D: Double digestion of 14-3-3γ; E: The monoclonal PCR identification results of positive recombinant 14-3-3γclones; F: The transfection efficiency of 14-3-3γ adenovirus vectors. Each experiment was repeated at least three times.
the cells were also affected by the over-expression of 14-3-3γ. The cells of both the Wt (blank control) group and the vector (negative control) group have similar morphology of elongated shapes (Fig. 2C). However, cells in 14-3-3γ over-expression group became rounded with less elongated cytoplasmic processes (Fig. 2C). Also there are more cells in 14-3-3γ over-expression group lost attachment to the plate, assumed smaller- and rounded-shape, a typical morphology of dying and degenerating cells. To examine whether over-expression of 14-3-3γ indeed affected cell death, apoptosis were assessed by flow cytometry and TUNEL assays. As shown in Fig. 2D and E, 14-3-3γ over-expression significantly increased cell apoptosis compared to Wt and vector groups (P < 0.01), suggesting that increase in apoptosis may also play a role in the differences in the number of cells maintained between 14-3-3γ and the two control groups (Fig. 2B). These results were also confirmed in the uterine leiomyoma immortal stem cells, which got the similar conclusions.
recombinant adenovirus (pHBAd-MCMV-RFP-14-3-3γ) or control vector (pHBAd-MCMV-RFP). Insertion of 14-3-3γ gene did not affect the infection efficiency (Fig. 1F). Infection of empty vector (pHBAd-MCMVRFP) did not change 14-3-3γ protein levels compared to the cells without treatment/infection (Wt), indicating that the virus and infection, by themselves, had no effect on the expression of 14-3-3γ (Fig. 2A). In contrast, infection of cells with 14-3-3γ-containing virus (pHBAd-MCMV-RFP-14-3-3γ), at 200 MOI, significantly increased the expression of the protein (Fig. 2A). 3.2. Effects of 14-3-3γ over-expression on cell proliferation and apoptosis For the cells received control vector, they increase in numbers during the first 36 h in culture and then slowly decrease in numbers through 60 h (Fig. 2B). However, for the cells over-expressing 14-3-3γ the numbers failed to increase during the first 24 h, which decreased sharply through 60 h, so by the end of culture, only half of the cells survived compared to the control group received empty vectors. Accompanied with the differences in the cell numbers, the morphology of 46
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Fig. 2. Effects of 14-3-3γ over-expression on human uterine leiomyoma cells proliferation and apoptosis. A: The expression of 14-3-3γ protein in cells transfected with 14-3-3γ overexpression; B: The effects of 14-3-3γ over-expression on cell proliferation; C: The effects of 14-3-3γ over-expression on cell morphology; D: The effects of 14-3-3γ over-expression on cell apoptosis by flow cytometry; E: The effects of 14-3-3γ over-expression on cell apoptosis by TUNEL assays. Each experiment was repeated at least three times.
The 42 differentially expressed proteins were analyzed by PANTHER database with different functional annotations (the molecular function, biological process, protein class, cell component, signaling pathway and others) (Fig. 3A). Cross-examinations of these annotations indicate that over-expression of 14-3-3γ has major effects on cellular proteins with binding/catalytic activities and proteins affecting the structure of the cells (Fig. 3A: Molecular function). Further classification indicates proteins involved in signaling and cytoskeletal/adhesion were among the ones affected the most (Fig. 3A: protein class). Finally, more detailed signaling pathway analysis has found that the affected signaling molecules concentrated on EGF-, FGF-, insulin/IGF-,
3.3. Effects of 14-3-3γ over-expression on the global changes of protein levels To elucidate the mechanism(s) by which the cell morphology, growth and apoptosis were affected by over-expression of 14-3-3γ, the global changes in protein levels were screened by Tandem Mass Tag combined with liquid chromatography-tandem mass spectrometry. Among the total of 5846 proteins detected, 42 proteins were found affected significantly by over-expression of 14-3-3γ (Table 1). In total, 9 proteins were significantly increased by at least 1.5 fold and 33 proteins were down-regulated significantly by at least 0.7 fold. 47
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pathway after 14-3-3γ over-expression, mTOR, TSC2 and Foxo1 proteins were examined because of their close relations to the AKT signaling pathway. As shown in Fig. 4B, 14-3-3γ over-expression significantly reduced the phosphorylations of AKT (p-AKT) and Foxo1 (pFoxo1), but not of mTOR (p-mTOR) or of TSC2 (p-TSC2). Also, 14-3-3γ over-expression did not affect the total protein levels of any of these 4 targets. Again, these results confirmed the above observations that 143-3γ affects the signaling pathways mostly by phosphorylation, not by synthesis/degradation of the protein themselves. Nuclear transportation of transcriptional factors is one of the critical steps for most of the signaling transduction processes. To examine whether 14-3-3γ over-expression could affect the cellular redistribution of the effecting factor Foxo1, nuclear and cytoplasm were isolated and the amounts of Foxo1 and p-Foxo1 were analyzed by Western blots in the two fractions (Fig. 5A). Relatively, the expression of both p-Foxo1 and Foxo1 were higher in nuclear than in cytoplasm for the vector and 14-3-3γ over-expression groups (P < 0.05). When the proteins were expressed as nuclear/cytoplasmic ratios, p-Foxo1 was significant reduced by 14-3-3γ over-expression (P < 0.01) compared to the vector group, while there was no significant difference of Foxo1 between the two groups (Fig. 5B, C). These results indicated that in addition to affect the overall phosphorylation of Foxo1, 14-3-3γ over-expression may also affect the distributions of p-Foxo1. It can specifically reduce the nuclear localization of pFoxo1. To further explore the possible changes in the upstream proteins of ERK1/2 pathway, phosphorylations of MEK1/2 were also examined (Fig. 6A). As shown in Fig. 6A, p-MEK1/2 and p-ERK1/2 were both down-regulated by 14-3-3γ over-expression (P < 0.01), while the total protein levels of MEK1/2 and ERK1/2 were not affected significantly, suggesting that loss of p-MEK1/2 may be the reason that p-ERK1/2 were reduced. In addition to the signaling molecules, two of the most important cell structure proteins, vimentin and desmin were also analyzed. Overexpression of 14-3-3γ did not affect the total protein levels of both vimentin and desmin, but significantly reduced the phosphorylated form of vimentin (p-Vimentin), but not desmin (Fig. 6B).
Table 1 The obvious changed protein when 14-3-3γ over-expression in uterine leiomyoma. Accession no.
Protein
Fold
Expression
P61981 P20591 P05362 P31947-2 O15078 P48307-2 P04179-4 P43490 A0A087WW59 F8WCR8 P49448 O75157-2 Q15582 Q04837 Q8IVF2 H3BM79 P17661 P31749 H7BY57 Q04917 Q8IZV5 O43524-2 Q12778 P09486 Q96B36-2 P29373 Q08AE8-2 Q15149-4 P31751 O14828-2 A0A087WW43 F5GXS0 B4DLN1 Q9BTT0-3 P08670 P27216 A0A0A0MT32 P01023 P02452 H0YN26 P04731 E9PND2
14-3-3 protein γ MX1 ICAM1 14-3-3 protein sigma CEP290 TFPI2 SOD2 NAMPT C11orf96 NCKIPSD GLUD2 TSC22D2 TGFBI SSBP1 AHNAK2 AKTIP Desmin AKT1 NFASC 14-3-3 protein eta RDH10 Foxo3 Foxo1 SPARC AKT1S1 CRABP2 SPIRE1 PLEC AKT2 SCAMP3 ITIH3 C4B Uncharacterized protein ANP32E Vimentin ANXA13 LIPA A2M COL1A1 ANP32A MT1A CSRP1
4.238 1.930 1.859 1.774 1.616 1.576 1.573 1.517 1.511 0.700 0.698 0.698 0.697 0.693 0.689 0.689 0.689 0.678 0.676 0.669 0.669 0.669 0.667 0.665 0.664 0.664 0.663 0.660 0.654 0.649 0.644 0.644 0.639 0.633 0.632 0.617 0.613 0.608 0.597 0.525 0.515 0.311
↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓
4. Discussion Despite the high prevalence of uterine leiomyomas in human population, which affects millions of woman around the world, their real cause remains unknown [1]. In the previous study, we have found that 14-3-3γ protein was down-regulated significantly in uterine leiomyoma compared to the surrounding normal myometrium [3]. We hypothesize that loss in 14-3-3γ protein may play a causal role in the growth/progression of uterine leiomyoma. To test this hypothesis, we have restored the loss of 14-3-3γ protein in uterine leiomyoma cells by overexpressing the protein through an adenoviral vector, which was constructed by our lab. Infection of uterine leiomyoma cells with the vector successfully resulted in significant increases in 14-3-3γ protein level in the cells. The transgenic approach largely normalized the level of 14-3-3γ in uterine leiomyoma cells to that of normal myometrium. Interestingly, restoration of 14-3-3γ significantly inhibited cell proliferation and promoted cell apoptosis. These results support our hypothesis that loss of 14-3-3γ may indeed play a causal role in the growth/progression of uterine leiomyoma. To elucidate the molecular mechanism involved, we have screened the changes in the protein levels, at globe scale, in the overexpressing cells and the phosphorylations of key members of a few well-known 143-3γ related signaling pathways. With the latest proteomics technology, 42 differentially expressed proteins were identified from a total of 5846 proteins examined. Bioinformatical analysis of the 42 differentially expressed proteins by PANTHER database and GeneMANIA, have revealed that the differentially regulated proteins are involved in variety biological processes, mostly in signaling transductions and structure
PI3- and chemokine/cytokine-pathways (Fig. 3A: signal pathway). To determine the interrelationship among these 42 genes, GeneMANIA network analysis was applied, the results of which are displayed in Fig. 3B. The derived network consisted of 22 of the 42 core genes and 14 additional genes that were pulled in by GeneMANIA. These results are consistent with the observations that 14-3-3γ functions as an adopt protein that is capable of modifying a broader range of signaling transductions.
3.4. Effects of 14-3-3γ over-expression on the signaling transductions of MAPK, AKT and Foxo1 To further examine how 14-3-3γ over-expression affect the key signaling pathways, which are potentially important to cell proliferation and apoptosis, phosphorylations of the key members of MAPK, AKT and Foxo1 were analyzed by phospho-MAPK pathway array and Western blots. As shown in Fig. 4A, over-expression of 14-3-3γ resulted in reductions in the phosphorylations of multiple members of the signaling molecules, including p-AKT1, p-AKT2, p-AKT pan, p-ERK1, p-GSK-3α and p-GSK-3β, while the protein themselves were not affected significantly, suggesting that 14-3-3γ affects the signaling pathways mostly by phosphorylation, not by synthesis/degradation of the protein themselves. These results were mostly confirmed by Western blots. To further explore the changes of the downstream proteins of AKT 48
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Fig. 3. The bioinformatics analysis of differentially expressed proteins after 14-3-3γ over-expression by PANTHER and GeneMANIA. A: The bioinformatics analysis of differentially expressed proteins after 14-3-3γ over-expression by PANTHER database with different functional annotations (the molecular function, biological process, protein class, cell component, signaling pathway and others); B: The interrelationship of differentially expressed proteins after 14-3-3γ over-expression by GeneMANIA network analysis.
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Fig. 4. Effects of 14-3-3γ over-expression on the signaling transductions of MAPK, AKT and Foxo1. A: Effects of 14-3-3γ over-expression on phospho-MAPK related proteins; B: Effects of 14-3-3γ over-expression on the expression of mTOR, TSC2, Foxo1 and AKT. Each experiment was repeated at least three times.
phosphorylation of ERK1/2. Such effect may result from the actions on the up-stream of the signaling pathway since accompanied by decrease in p-ERK1/2, p-MEK1/2 was also reduced by over-expression of 14-33γ. At present, however, it is not clear how 14-3-3γ inhibits MEK1/2 phosphorylations. Overall, the results suggest that inhibition of MEK1/ 2-ERK1/2 signaling could be one of the mechanisms by which 14-3-3γ inhibits uterine leiomyoma cell proliferation and induces their apoptosis. This conclusion is also supported by findings in breast epithelial cells, where 14-3-3γ could reduce the cellular damage caused by lipopolysaccharide through inhibiting the ERK1/2 signaling [25]. In addition to MAPK, AKT is another signaling pathway that plays a central role in regulating cell survival, proliferation, apoptosis and migration. Evidences suggest that activation of this signaling may also play a role in the growth/progression of uterine leiomyoma. For example, Xu et al. [26] found that the expression of p-AKT in uterine leiomyoma was significantly increased compared to that in surrounding normal myometrium tissues, and that blocking the signaling in uterine leiomyoma cells significantly increased cell apoptosis. Similarly, inhibition of PI3K (an up-stream kinase that phosphorylates AKT) can increase uterine leiomyoma cell apoptosis and promote cell cycle arrested [27]. Although the involvement of AKT signaling in the growth/ progression of uterine leiomyoma seems clear, the mechanisms/factors that increase AKT and/or its phosphorylation is still unknown. In our
regulations. However, these changes in the quantity of proteins, by themselves, cannot explain the overall changes (proliferation, apoptosis and morphology) observed in the cells. To elucidate more detailed mechanisms involved, the phosphorylation of the two major signaling pathways, MAPK and AKT, have been examined. These pathways were selected for three reasons: 1) They are closely related to cell proliferation and apoptosis; 2) They are among the most common pathways modified by 14-3-3γ; 3) They are frequently modified by treatments/manipulations that affected uterine leiomyoma cell proliferation and/or apoptosis. ERK1/2 is one of the best studied MAPK signaling pathway members (Ras/Raf/MEK/ERK pathway). It is well known that this pathway plays significant roles in the regulation of cell proliferation and apoptosis [21,22]. Chegini et al. reported that the expression of ERK1/2 protein was increased in uterine leiomyoma, compared to the surrounding normal myometrium [23]. Such increase most likely plays a causal role in uterine leiomyoma growth since inhibition of the signaling (MEK1/2-ERK1/2) significantly reduced the proliferation of uterine leiomyoma cells [10,23,24]. However, the mechanism that is responsible for the up-regulation of ERK1/2 or its phosphorylation is still unknown. In the present study, we have found that the loss of 14-33γ may play a role in the increase of p-ERK1/2 in uterine leiomyoma cells since increase/restoration of 14-3-3γ significantly reduced the
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Fig. 5. Effects of 14-3-3γ over-expression on the nuclear and cytoplasmic expression of Foxo1 protein. A: Effects of 14-3-3γ over-expression on the nuclear and cytoplasmic expression of p-Foxo1 and Foxo1 proteins by Western blot; B: Effects of 14-3-3γ over-expression on the nuclear and cytoplasmic expression of p-Foxo1 and Foxo1 proteins; C: Effects of 14-3-3γ overexpression on the nuclear/cytoplasmic expression of p-Foxo1 and Foxo1 proteins. Each experiment was repeated at least three times.
has been frequently observed in other systems. For example, it is well known that 14-3-3 protein can carry its physiological function and keep the dynamic balance of cell apoptosis through affecting the phosphorylation of AKT at threonine 308 site [28]. In human breast cancer cells, 14-3-3σ up-regulation could inhibit cell proliferation and tumor
present study, we have found that loss of 14-3-3γ may play a causal role in the activation of AKT signaling since increase/restoration of 14-3-3γ in uterine leiomyoma cells significantly reduced the phosphorylation of AKT and its down-stream effectors. The regulatory relationship between 14-3-3γ and AKT is not special to uterine leiomyoma cells since it
Fig. 6. Effects of 14-3-3γ over-expression on the expression of selected potential function proteins. A: Effects of 14-3-3γ over-expression on the expression of p-MEK1/2, MEK1/2, p-ERK1/ 2 and ERK1/2 proteins by Western blot; B: Effects of 14-3-3γ over-expression on the TGFBI, p-Vimentin, Vimentin and Desmin proteins by Western blot. Each experiment was repeated at least three times.
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progression of human uterine leiomyoma. Compared to previous studies, there are some new discoveries in this manuscript. Increase/restoration of 14-3-3γ in uterine leiomyomal cells reduced cell proliferation and increased cell apoptosis. AKT-Foxo, MEK1/2-ERK1/2 and vimentin signaling may be involved, while TSC2, mTOR, TGFBI and Desmin are unlikely to play any significant role in the process. The mechanism that is responsible for the loss of 14-3-3γ in human uterine leiomyoma, however, is still unknown and deserves for further study.
progression via inhibiting AKT activation [29]. In the breast epithelial cells, 14-3-3γ up-regulation can reduce the cell damage induced by lipopolysaccharide through the activation of AKT pathway [25]. Foxo, the downstream effecting molecule of AKT pathway, often functions as a tumor suppressor. Its function can be negatively regulated by Akt-mediated phosphorylation, since p-Foxo is often transported from nucleus into cytoplasm, where it is bound with 14-3-3 protein, and prevented from binding to target gene transcriptions [30]. Kovács et al. [31] have found that the expressions of p-AKT and pFoxo1 were both increased in uterine leiomyoma, and that the p-Foxo1 in the nucleus was significantly higher in leiomyoma than in normal myometrium. Foxo is a 14-3-3 protein ligand, through which 14-3-3 protein can regulate nucleus/cytoplasm distribution of Foxo [6]. Also, 14-3-3 protein can affect cell proliferation and apoptosis by preventing the Foxo from degradation or dephosphorylation [12]. In this study, we have found that both p-AKT and p-Foxo was significantly inhibited by over-expression of 14-3-3γ, and p-Foxo nucleus/cytoplasm expression ratio was also affected (decreased). We found that 14-3-3γ could not only decrease the overall phosphorylation of Foxo1, but also reduce the nuclear localization of p-Foxo1. This mechanism may be that over-expression of 14-3-3γ firstly decreased the total p-AKT, then total p-Foxo was decreased; 14-3-3γ also regulated the p-Foxo transportation from nucleus into cytoplasm. These changes relatively increased the level of Foxo, which functions as a tumor suppressor. These observations suggest strongly that p-AKT/p-Foxo down-regulation could be one of the likely reasons that over-expression of 14-3-3γ resulted in increasing cell apoptosis and decreasing cell proliferation in uterine leiomyoma. Vimentin is a member of the intermediate filament family, which in addition to maintain the integrity of the cell, can involve in cell migration, adhesion, signal transduction, and cell apoptosis [32]. Vimentin is expressed by a variety of tumor cells and may be served as a marker for assessing the degrees of cancer malignancy [33]. Vimentin can regulate 14-3-3 activities by binding to the protein when phosphorylated, which inhibits the ability of 14-3-3 binding to other targets [34]. Vimentin, AKT and 14-3-3 can form a complex to participate in tumor cell autophagy and apoptosis. Also, vimentin can be regulated by the ERK1/2 pathway [35]. We have found that the expression of vimentin was also increased in uterine leiomyoma compared to the surrounding normal myometrium. In the present study, the decrease in pVimentin protein by over-expression 14-3-3γ suggests that vimentin could be another player in the promotions of growth/progression of uterine leiomyoma cells by loss of 14-3-3γ. In addition to Foxo1 and vimentin, this study also examined other potential targets and/or modifying members of AKT and/or ERK1/2 signaling. These include: TSC2, a tumor suppressor, locating at the downstream of the AKT pathway [36]; mTOR, another important downstream target of AKT [37,38], which also was up-regulated in uterine leiomyomas [39]; TGFBI, an important regulator in tumorigenesis of a broader range of systems [40], which was also up-regulated in uterine leiomyoma [3]; and Desmin, a cytoskeletal protein, often being used as one of the markers of cell proliferation and differentiation in early stage [41], which was also up-regulated in uterine leiomyoma [3]. Although most of these potential target proteins were increased in uterine leiomyoma, over-expression of 14-3-3γ did not significantly affect the levels of the total proteins or phosphorylations, suggesting that they are unlikely the targets and/or mediators in 14-3-3γ induced down-regulation of cell apoptosis and up-regulation of cell divisions. Besides, in our previous study [42], we have found that 14-3-3γ promoter was methylated more in leiomyomas than in matched myometrium, and 14-3-3γ was negatively correlated with ER and PR proteins, whose expressions were higher in leiomyomas. These results may suggest that the hypermethylation of the 14-3-3γ promoter may account, at least partially, for the down-regulation of 14-3-3γ in leiomyomas. In summary, 14-3-3γ expression is reduced in human uterine leiomyoma, which may be a causal factor in the development and/or
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