April 2011
Volume 52, Issue 5
Free
Biochemistry and Molecular Biology  |   April 2011
TGF-β Induces Sustained Upregulation of SNAI1 and SNAI2 through Smad and Non-Smad Pathways in a Human Corneal Epithelial Cell Line
Author Affiliations & Notes
  • Keiichi Aomatsu
    From the Departments of Ophthalmology and
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Tokuzo Arao
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Koji Sugioka
    From the Departments of Ophthalmology and
  • Kazuko Matsumoto
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Daisuke Tamura
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Kanae Kudo
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Hiroyasu Kaneda
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Kaoru Tanaka
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Yoshihiko Fujita
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Yoshikazu Shimomura
    From the Departments of Ophthalmology and
  • Kazuto Nishio
    Genome Biology, Kinki University School of Medicine, Osaka, Japan.
  • Corresponding author: Kazuto Nishio, Department of Genome Biology, Kinki University School of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511, Japan; knishio@med.kindai.ac.jp
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2437-2443. doi:10.1167/iovs.10-5635
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      Keiichi Aomatsu, Tokuzo Arao, Koji Sugioka, Kazuko Matsumoto, Daisuke Tamura, Kanae Kudo, Hiroyasu Kaneda, Kaoru Tanaka, Yoshihiko Fujita, Yoshikazu Shimomura, Kazuto Nishio; TGF-β Induces Sustained Upregulation of SNAI1 and SNAI2 through Smad and Non-Smad Pathways in a Human Corneal Epithelial Cell Line. Invest. Ophthalmol. Vis. Sci. 2011;52(5):2437-2443. doi: 10.1167/iovs.10-5635.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The aim of this study was to investigate the expression changes of epithelial mesenchymal transition (EMT)-related molecules induced by TGF-β signaling in a human corneal epithelial cell line (HCECs).

Methods.: The cellular response to TGF-β was evaluated by immunoblotting, quantitative real-time RT-PCR, and immunofluorescence microscopy in HCECs.

Results.: TGF-β significantly increased mRNA expression of SNAI1, SNAI2, VIM, and FN1, but not TWIST1 through Smad and non-Smad pathways in HCECs. Protein expression of a mesenchymal marker N-cadherin was dose-dependently increased and that of an epithelial marker of E-cadherin was decreased by TGF-β. TGF-β, but not EGF, mediated the EMT-like morphologic changes. Both TGF-β and EGF were capable of upregulating SNAI1 and SNAI2 by about two-fold within a short response time. However, a detailed time course analysis revealed drastically different expression patterns, with TGF-β mediating a sustained upregulation of SNAI1 and SNAI2 for at least for 6 days and EGF allowing a return to the baseline expression values after 8 ∼ 12 h. These data indicate that TGF-β, but not EGF, induces sustained upregulation of SNAI1 and SNAI2 in HCECs.

Conclusions.: TGF-β induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways, EMT-like morphologic changes, downregulation of E-cadherin, and upregulation of N-cadherin in HCECs. The authors' findings provide insight into the TGF-β signaling and the temporal expression patterns of EMT-inducible transcription factors in HCECs.

TGF-β is a multipotent growth factor that can exert multiple functions including the induction of cell proliferation, differentiation, cell cycle arrest, apoptosis, and/or transformation in time- and system-dependent manners by binding to transmembrane serine/threonine kinase receptors. 1,2 TGF-β is well-known as a potent initiator of epithelial mesenchymal transition (EMT) and activates several transcription factors to induce EMT via Smad or non-Smad pathways. 1 These EMT-inducible transcription factors are known as zinc finger factors (Snail, Slug, EF1, and SIP1) and basic helix-loop-helix factors (Twist, E2A, ID2/3, and E12/E47). 1 Among the non-Smad signaling responses, activation of extracellular signal-regulated kinases (ERKs), Rho GTPases, and the PI3 kinase/AKT pathway in response to TGF-β have been linked to TGF-β–induced EMT through their regulation of distinct processes, such as cytoskeleton organization, cell growth, survival, migration, or invasion. 3 TGF-β also activates p38 MAP kinase and induces EMT through p38 MAP kinase in NMuMG cells. 4 On the other hand, EGF receptor (EGFR) activation enhances the EMT response in renal tubular epithelial cells, 5 and EGFR cooperates with integrin signaling to induce EMT via the upregulation of SNAI1 gene expression in cervical cancer cells. 6 The EGF/EGFR signaling pathways can also induce cancer cell EMT via STAT3-mediated TWIST gene expression. 7  
In the corneal epithelial cells, TGF-β enhances cellular migration and inhibits cellular proliferation in corneal epithelial cells in vitro and in vivo. 8 During the wound healing of corneal epithelium, TGF-β is upregulated and the corneal epithelial cells migrate into the injured area. The cells lack cellular proliferation in early phase, but begin to proliferate when epithelial defect is recovered. 9 The corneal epithelial cells undergo phenotypic changes to gain migratory characteristics in a way similar to the EMT process through activation of the p38 MAPK cascade during wound healing. 10 Regarding TGF-β receptors, TGF-β receptor-I and TGF-β receptor-II are both upregulated in cells migrating to cover a corneal wound after wounding. 11 Collectively, TGF-β signaling is considered to play a critical role in corneal wound healing. 
Notably, a recent report has demonstrated that the expression levels of Slug/SNAI2, a member of the Snail family of EMT regulator, was upregulated at sites of epithelial cell migration at the margins of normally healing corneal wounds, while did not occur at the margins of non-healing corneal erosions. 12 Thus, the investigation of the TGF-β-mediated phenotypes in corneal epithelial cells may be valuable to understand the corneal cell biology in wound healing. In this study, we investigated the expression changes of EMT-related molecules in human corneal epithelial cells (HCECs). 
Methods
Cell Cultures
The Sv40-immortalized human corneal epithelial cell (HCEC) line was used in this study. 13 HCECs were maintained in DMEM/F12 medium (Gibco, BRL, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS; Gibco) and gentamicin (40 μg/mL; Sigma, St. Louis, MO) at 37°C in a humidified incubator with 5% CO2. The A549 cell line was cultured in RPMI-1640 medium (Sigma) with 10% FBS. 
Reagents
Human TGF-β and EGF were purchased from R&D Systems (Minneapolis, MN). SB431542 (Sigma) and AG1478 (Biomol, Inc., St. Louis, MO), U0126 and Wortmannin (Cell Signaling Technology, Beverly, MA) were dissolved in dimethylsulfoxide for stock solution. 
Western Blot Analysis
The antibodies used in this study were anti-Smad2, anti-phospho-Smad2, anti-EGFR, anti-phospho EGFR, anti-Akt, anti-phospho Akt, anti-MAPK, anti-phospho MAPK, anti-snail, anti-β actin, HRP-conjugated secondary antibody (Cell Signaling), anti-E cadherin, and anti-N cadherin (Invitrogen, San Diego, CA). A western blot analysis was performed as described previously. 14 The western blot experiments for evaluating signal transduction were performed under serum-starved conditions (see Figs. 1A, 3A, 5A–D, 6D, and 6E), while the other experiments were performed in the presence of 15% serum because of the cellular damage that occurs as a result of serum-starved conditions for long periods of time. The densitometry analysis was performed using commercial software (MultiGauge ver. 3.1 Fujifilm, Tokyo, Japan). The experiment was performed in triplicate. 
Cell Proliferation Assay
Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously. 15 The experiment was performed in triplicate. 
Real-Time Reverse Transcription–PCR
The real-time RT-PCR method was previously described. 16 The following primers were used: SNAI1, forward 5′-TCT AGG CCC TGG CTG CTA CAA-3′ and reverse 5′-ACA TCT GAG TGG GTC TGG AGG TG-3′; SNAI2, forward 5′-ATG CAT ATT CGG ACC CAC ACA TTA C-3′ and reverse 5′-AGA TTT GAC CTG TCT GCA AAT GCT C-3′; TWIST1, forward 5′-GCC TTC TCG GTC TGG AGG AT-3′ and reverse 5′-TTT CTC CTT CTC TGG AAA CAA TGA C-3′; vimentin, forward 5′-TGA GTA CCG GAG ACA GGT GCA G-3′ and reverse 5′-TAG CAG CTT CAA CGG CAA AGT TC-3′; fibronectin, forward 5′-GAG CTG CAC ATG TCT TGG GAA C-3′ and reverse 5′-GGA GCA AAT GGC ACC GAG ATA-3′; and GAPD, forward 5′-GCA CCG TCA AGG CTG AGA AC-3′ and reverse 5′-ATG GTG GTG AAG ACG CCA GT-3′. GAPD was used to normalize the expression levels in the subsequent quantitative analyses. 
Phase-Contrast Microscopy
The HCECs were cultured on 6-well plates. Morphologic changes were detected and photographed using immunofluorescence microscopy (IX71-PAFM; Olympus, Tokyo, Japan) after stimulation with a growth factor for 72 h. The percentage of spindle-shape cells was determined for each view. 
Fluorescence Imaging
HCECs cultured on coverslips were fixed with 4% paraformaldehyde for 15 minutes at 37°C, then washed three times with PBS and blocked in 5% goat serum (DakoCytomation, Glostrup, Denmark) in PBS. The cells were further incubated with primary antibody, anti-E cadherin and anti-N cadherin (1:500) (Invitrogen) for 1 hour, secondary Alexa Fluor 488–conjugated goat anti-mouse IgG antibody (Invitrogen) for 1 hour at room temperature. For the detection of F-actin rearrangement, the cells were fixed with 4% paraformaldehyde for 15 minutes at 37°C, then washed three times with PBS and permeabilized by a 5-minute incubation with TBS solution (0.1% Triton X-100/1% BSA/PBS). The coverslips were again washed three times in PBS and immunostained with Rhodamine-conjugated Phalloidin (Invitrogen) for 15 minutes at room temperature. The nuclei were subsequently stained for 5 minutes with 4′,6-diamidino-2-phenylindole (DAPI). E-cadherin, N-cadherin, Rhodamine, and DAPI fluorescence were detected using immunofluorescence microscopy IX71-PAFM (Olympus) and E800 (Nikon, Tokyo, Japan). 
Statistics
The statistical analyses were performed using commercial software (Excel; Microsoft, Redmond, WA) to calculate the average ± SD and to test for statistically significant differences between the samples using a Student's t-test. A P-value < 0.05 was considered statistically significant. 
Results
TGF-β Upregulated SNAI1 and SNAI2 Expressions in HCECs
TGF-β increased the phosphorylation levels of Smad2 in a dose-dependent manner, and the effect was observed within 15 minutes after stimulation (Fig. 1A). Generally, TGF-β is a potent growth inhibitor of epithelial cells in vitro. In line with this feature, TGF-β significantly inhibited the cell proliferation of HCECs in a dose-dependent manner (Fig. 1B). This result indicates that TGF-β signaling actually provides a signal response to HCECs. 
Figure 1.
 
TGF-β signaling actually provides a signal response to human corneal epithelial cells (HCECs). (A) Western blot examining the phosphorylation and expression levels of Smad2 in time- (0, 15, 30, and 60 min of TGF-β stimulation) and dose- (0.1, 1, and 10 ng/mL) dependent responses to TGF-β stimuli in HCECs. β-actin was used as an internal control. Marker, protein size marker. (B) Growth inhibitory effect of TGF-β in HCECs evaluated using an MTT assay after incubation for 0, 24, 48, and 72 h. The experiments were performed in triplicate. The error bars represent the SD. *P < 0.05.
Figure 1.
 
TGF-β signaling actually provides a signal response to human corneal epithelial cells (HCECs). (A) Western blot examining the phosphorylation and expression levels of Smad2 in time- (0, 15, 30, and 60 min of TGF-β stimulation) and dose- (0.1, 1, and 10 ng/mL) dependent responses to TGF-β stimuli in HCECs. β-actin was used as an internal control. Marker, protein size marker. (B) Growth inhibitory effect of TGF-β in HCECs evaluated using an MTT assay after incubation for 0, 24, 48, and 72 h. The experiments were performed in triplicate. The error bars represent the SD. *P < 0.05.
Next, we examined the induction of EMT-inducible transcription factors by TGF-β stimuli. TGF-β upregulated the mRNA expression of SNAI1 and SNAI2, which are key EMT regulators, but the expression of TWIST1 was not changed (Fig. 2A). The mRNA expression of vimentin and fibronectin were upregulated (Fig. 2A). 
Figure 2.
 
TGF-β upregulates EMT-related molecules via Smad and non-Smad pathways in HCECs. (A) The mRNA expression levels of SNAI1, SNAI2, TWIST1, VIM (vimentin), and FN1 (fibronectin 1) were determined using a real-time RT-PCR analysis. HCECs were stimulated with TGF-β at 0.1, 1, and 10 ng/mL for 24 h and the cells were used for analysis. (B) Real-time RT-PCR analysis for mRNA expression levels of SNAI1 and SNAI2 via Smad or non-Smad signaling. HCECs were cultured with or without 10 μM of the MEK inhibitor U0126, 1 μM of the phosphoinositide-3-kinase inhibitor Wortmannin, or 10 μM of the TGF-β receptor inhibitor SB431542 for 30 min and were stimulated with 10 ng/mL of TGF-β for 8 h; the cells were then collected for analysis. Rel mRNA, normalized mRNA expression levels (target gene/GAPD × 103). The experiments were performed in triplicate. The error bars represent the SD. *P < 0.05.
Figure 2.
 
TGF-β upregulates EMT-related molecules via Smad and non-Smad pathways in HCECs. (A) The mRNA expression levels of SNAI1, SNAI2, TWIST1, VIM (vimentin), and FN1 (fibronectin 1) were determined using a real-time RT-PCR analysis. HCECs were stimulated with TGF-β at 0.1, 1, and 10 ng/mL for 24 h and the cells were used for analysis. (B) Real-time RT-PCR analysis for mRNA expression levels of SNAI1 and SNAI2 via Smad or non-Smad signaling. HCECs were cultured with or without 10 μM of the MEK inhibitor U0126, 1 μM of the phosphoinositide-3-kinase inhibitor Wortmannin, or 10 μM of the TGF-β receptor inhibitor SB431542 for 30 min and were stimulated with 10 ng/mL of TGF-β for 8 h; the cells were then collected for analysis. Rel mRNA, normalized mRNA expression levels (target gene/GAPD × 103). The experiments were performed in triplicate. The error bars represent the SD. *P < 0.05.
Smad and Non-Smad Pathways Were Involved in TGF-β–Mediated Upregulation of SNAI1 and SNAI2
Both Smad-mediated and non-Smad–mediated (ERK or AKT) pathways are involved in TGF-β–mediated EMT 17 ; therefore, we examined the downstream signal pathway in HCECs. The mRNA expression levels of SNAI1 and SNAI2 were upregulated by TGF-β by about two-fold (Fig. 2B). Half of the level of upregulation was effectively canceled by 10 μM of the MEK inhibitor U0126 or the phosphoinositide-3-kinase inhibitor Wortmannin, and the upregulations of SNAI1 and SNAI2 were completely canceled by 10 μM of the TGF-β receptor inhibitor SB431542 (Fig. 2B). The expression of TWIST1 was apparently unchanged. In TGF-β–dependent responses, these data indicated that both Smad and non-Smad pathways were certainly involved in TGF-β–mediated EMT in HCECs. 
TGF-β Downregulated E-cadherin and Upregulated N-cadherin in HCECs
Western blotting revealed that the protein expression of a mesenchymal marker N-cadherin was dose-dependently increased and epithelial marker of E-cadherin was slightly decreased by TGF-β at 24∼48 h after ligand stimulation in HCEC (Fig. 3A). The expression level of E-cadherin was clearly decreased and N-cadherin was increased in long-term treatment of TGF-β at 4∼6 days (Fig. 3B). In addition, HCECs expressed the epithelium-specific marker keratin 12 at baseline, and TGF-β dose-dependently downregulated the expression of this marker (Supplementary Fig. S1A). Western blotting and immunocytochemistry revealed that TGF-β also upregulated the protein expressions of vimentin and fibronectin (Supplementary Fig. S1B). Immunofluorescence analysis also showed that TGF-β decreased the expression of E-cadherin on cellular membrane and increased N-cadherin (Fig. 3C). 
Figure 3.
 
Protein expression regulation of N-cadherin and E-cadherin by TGF-β. (A) Western blotting for N-cadherin and E-cadherin were examined in HCECs and the EMT-positive control A549, a lung cancer cell line. HCECs were stimulated with the indicated concentration of TGF-β and the cells were collected at 24 h and 48 h. β-Actin was used as an internal control. Marker, protein size marker. (B) Western blotting for N-cadherin and E-cadherin were examined in HCECs for long time exposure. HCECs were stimulated with TGF-β at 10 ng/mL without serum starvation at indicated duration (4–6 days). β-actin was used as an internal control. (C) Immunofluorescent staining of HCECs treated with TGF-β (10 ng/mL) or without (Cont.) for 6 days. HCECs were stained for N-cadherin (upper panels) and E-cadherin (lower panels) and the nuclei of cells were counterstained with DAPI. Scale bar, 25 μm.
Figure 3.
 
Protein expression regulation of N-cadherin and E-cadherin by TGF-β. (A) Western blotting for N-cadherin and E-cadherin were examined in HCECs and the EMT-positive control A549, a lung cancer cell line. HCECs were stimulated with the indicated concentration of TGF-β and the cells were collected at 24 h and 48 h. β-Actin was used as an internal control. Marker, protein size marker. (B) Western blotting for N-cadherin and E-cadherin were examined in HCECs for long time exposure. HCECs were stimulated with TGF-β at 10 ng/mL without serum starvation at indicated duration (4–6 days). β-actin was used as an internal control. (C) Immunofluorescent staining of HCECs treated with TGF-β (10 ng/mL) or without (Cont.) for 6 days. HCECs were stained for N-cadherin (upper panels) and E-cadherin (lower panels) and the nuclei of cells were counterstained with DAPI. Scale bar, 25 μm.
TGF-β Mediated EMT-like Morphologic Changes in HCECs
From a morphologic aspect, EMT is characterized by an increase in scattering and an elongation of the cell shape. 18 TGF-β mediated both cell scattering and the elongation of cell-shape in HCECs, whereas no effect was observed with EGF, which is also known to be an EMT-inducible ligand (Figs. 4A, 4B). Phalloidin/DAPI staining for the visualization of intracellular F-actin enabled the rearrangement of the cytoskeletal organization from cell-cell borders into stress fibers in response to TGF-β, but not EGF, to be detected (Fig. 4A). The data showed that TGF-β, but not EGF, mediates EMT-like morphologic changes in HCECs. 
Figure 4.
 
TGF-β, but not EGF, mediates EMT-like morphologic changes in HCECs. (A) Morphologic changes were evaluated using phase-contrast microscopy (upper panels) and phalloidin and DAPI staining under fluorescence microscopy (middle and lower panels). HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for 72 h and then evaluated. Scale bar: (middle panels) 100 μm; (lower panels) 25 μm. (B) The ratio of spindle-shaped cells was then analyzed (ratio of cells with twofold > length/width in a high power field). Five random fields per sample at a magnification of ×200 were captured and evaluated. The error bars represent the SD. *P < 0.05.
Figure 4.
 
TGF-β, but not EGF, mediates EMT-like morphologic changes in HCECs. (A) Morphologic changes were evaluated using phase-contrast microscopy (upper panels) and phalloidin and DAPI staining under fluorescence microscopy (middle and lower panels). HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for 72 h and then evaluated. Scale bar: (middle panels) 100 μm; (lower panels) 25 μm. (B) The ratio of spindle-shaped cells was then analyzed (ratio of cells with twofold > length/width in a high power field). Five random fields per sample at a magnification of ×200 were captured and evaluated. The error bars represent the SD. *P < 0.05.
Inactive Crosstalk between TGF-β and EGF Signals in HCECs
Since crosstalk between the EGFR-ERK and TGF-β-Smad signaling pathways is known to enhance TGF-β–dependent responses, 19,20 we evaluated the crosstalk. The phosphorylation level of Smad2 increased in response to TGF-β stimulation and was canceled by SB431542; however, EGF did not increase the level of phosphorylation (Fig. 5A). On the contrary, EGF markedly increased the phosphorylation levels of EGFR, ERK and AKT, while TGF-β slightly increased the p-ERK and p-AKT levels (Fig. 5B). When both the TGF-β and EGF signals were simultaneously activated, no increase in the phosphorylation of Smad2 over EGF alone was noted (Fig. 5C). The p-ERK level, but not the p-EGFR or p-AKT levels, increased slightly with the addition of TGF-β (Fig. 5D). These results indicate that there is no detectable crosstalk between TGF-β and EGF in HCECs. 
Figure 5.
 
Inactive crosstalk between TGF-β and EGF signals in HCECs. Western blot for phospho-Smad2 and Smad2 expression (A, C) and phospho-EGFR, -ERK, and -AKT and these protein expression levels (B, D) are shown. HCECs were cultured with or without exposure to SB431542 or the EGFR tyrosine kinase inhibitor AG1478 for 30 min and stimulated with 10 ng/mL of TGF-β or EGF for 1 h or 15 min, respectively (A, B). For simultaneous stimulation with TGF-β and EGF, the HCECs were cultured with or without 10 μM of U0126, 1 μM of Wortmannin, or 1 μM of AG1478 for 30 min and were then stimulated with 10 ng/mL of TGF-β and EGF for 1 h or 15 min, respectively (C, D). β-Actin was used as an internal control. The experiments were performed in duplicate. Marker, protein size marker.
Figure 5.
 
Inactive crosstalk between TGF-β and EGF signals in HCECs. Western blot for phospho-Smad2 and Smad2 expression (A, C) and phospho-EGFR, -ERK, and -AKT and these protein expression levels (B, D) are shown. HCECs were cultured with or without exposure to SB431542 or the EGFR tyrosine kinase inhibitor AG1478 for 30 min and stimulated with 10 ng/mL of TGF-β or EGF for 1 h or 15 min, respectively (A, B). For simultaneous stimulation with TGF-β and EGF, the HCECs were cultured with or without 10 μM of U0126, 1 μM of Wortmannin, or 1 μM of AG1478 for 30 min and were then stimulated with 10 ng/mL of TGF-β and EGF for 1 h or 15 min, respectively (C, D). β-Actin was used as an internal control. The experiments were performed in duplicate. Marker, protein size marker.
Sustained SNAI1 and SNAI2 Upregulation Induced by TGF-β, but Not EGF
TGF-β, but not EGF mediated the morphologic changes in HCECs (Figs. 4A. 4B); therefore we focused on the temporal regulation of SNAI1 and SNAI2 expression to explain these differences. Notably, both TGF-β and also EGF significantly upregulated the mRNA expressions of SNAI1 and SNAI2 by about two-fold at 2 h after ligand stimulation (Fig. 6A). However, a detailed time course analysis revealed that the upregulation of SNAI1 expression induced by EGF was immediately restored to the baseline level within a short time period (∼8 h) (Fig. 6B). Interestingly, TGF-β induced the sustained upregulation of SNAI1 and SNAI2 for >48 h (Fig. 6B), and further analysis showed that the upregulation of SNAI1 and SNAI2 was sustained for at least 6 days (Fig. 6C). These findings indicate that the temporal expression regulation of SNAI1 and SNAI2 by TGF-β or EGF occurs in very different manners. Western blotting showed that both TGF-β and EGF increased the short-term protein expression of SNAI1 at 8 h; however, only TGF-β induced the sustained expression of SNAI1 at 24 h thereafter (Fig. 6C, 6D), indicating that the present data were consistent with the results of real-time RT-PCR. 
Figure 6.
 
TGF-β, but not EGF, induced the sustained upregulation of SNAI1 and SNAI2. (A) The mRNA expression levels of SNAI1 and SNAI2 were determined using a real-time RT-PCR analysis. HCECs were stimulated with 10 ng/mL of TGF-β or EGF for 2 h and the cells were analyzed. (B) Detailed time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-β or EGF. HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for the indicated time course. Note that TGF-β resulted in the sustained upregulation of SNAI1 and SNAI2 for >48 h. Rel mRNA, normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. (C) Further time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-β. HCECs were cultured in the presence of 10 ng/mL of TGF-β for the indicated time course. Normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. *P < 0.05. (D) Western blotting for SNAI1/Snail1. HCECs were cultured in the presence of 10 ng/mL of TGF-β for the indicated time course and exposed to 10 μM of SB431542 at 8 h. β-actin was used as an internal control. M, protein size marker. (E) Long-term expression of SNAI1 by TGF-β or EGF. HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for the indicated time course. β-actin was used as an internal control.
Figure 6.
 
TGF-β, but not EGF, induced the sustained upregulation of SNAI1 and SNAI2. (A) The mRNA expression levels of SNAI1 and SNAI2 were determined using a real-time RT-PCR analysis. HCECs were stimulated with 10 ng/mL of TGF-β or EGF for 2 h and the cells were analyzed. (B) Detailed time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-β or EGF. HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for the indicated time course. Note that TGF-β resulted in the sustained upregulation of SNAI1 and SNAI2 for >48 h. Rel mRNA, normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. (C) Further time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-β. HCECs were cultured in the presence of 10 ng/mL of TGF-β for the indicated time course. Normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. *P < 0.05. (D) Western blotting for SNAI1/Snail1. HCECs were cultured in the presence of 10 ng/mL of TGF-β for the indicated time course and exposed to 10 μM of SB431542 at 8 h. β-actin was used as an internal control. M, protein size marker. (E) Long-term expression of SNAI1 by TGF-β or EGF. HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for the indicated time course. β-actin was used as an internal control.
Discussion
TGF-β induced sustained upregulation of SNAI1 and SNAI2 in HCECs. Lu et al. 21 clearly demonstrated that EGF induced EMT and the sustained upregulation of SNAI1 for up to 5 days in A431 cell lines, and their data are consistent with the findings of the present study. Although emerging data indicates that SNAI1 activity is regulated via post-transcriptional regulatory mechanisms, such as its phosphorylation by GSK3—which leads to its inhibition by ubiquitination and degradation, 22 our results provide another regulatory mechanism of SNAI1 expression at a transcriptional level. Meanwhile, the upregulation of TWIST1 by TGF-β is known to occur during the process of chondrocyte progression toward terminal maturation 23 and during EMT via HMGA2 24 in a physiological context. In the corneal epithelial cells, TGF-β did not upregulate TWIST1 expression. This result suggests that TWIST1 is not likely to be induced by TGF-β signaling in HCECs. 
Many studies have demonstrated crosstalk between TGF-β- and EGF-stimulated pathways through the facilitation of EMT. For example, the phosphorylation of the ERK-dependent R-Smad linker region enhances collagen I synthesis, implying positive crosstalk between the ERK and Smad pathways in human mesangial cells. 19 EGF plus TGF-β induced a dramatic morphologic change characteristic of EMT in rat intestinal epithelial (RIE) cells, and TGF-β augmented the EGF-mediated signaling of ERK and AKT by enhancing and prolonging the activation of the former and prolonging the activation of the latter. 20 EGF and TGF-β synergistically stimulated proximal tubular cell migration after EMT through an increase in MMP-9 function and enhanced ERK1/2 activation. 25 Unexpectedly, the activation of the Smad pathway stimulated by the EGF signal was not seen in this study, indicating that no detectable crosstalk exists between the TGF-β and EGF signal pathways in HCECs. 
In general, EMT is a cellular process with a dramatic remodeling of the cytoskeleton and a losing of cell-cell contacts. 26 A hallmark of EMT is the loss of E-cadherin expression and the cells undergoing EMT acquire expression of mesenchymal components and manifest a migratory phenotype. 27 We found that TGF-β induced sustained upregulation of SNAI1 and SNAI2 via both Smad and non-Smad pathways in HCECs (Fig. 7). Furthermore, TGF-β mediated EMT-like morphologic changes, downregulated E-cadherin, and upregulated N-cadherin in HCECs. Although further in vivo studies are necessary, these results suggest that TGF-β mediates the cellular phenotype toward EMT in HCECs. Taken together, our findings provide insight into the TGF-β signaling and the temporal expression patterns of EMT-inducible transcription factors in HCECs. 
Figure 7.
 
Proposed model depicting the signaling pathway of TGF-β–mediated expressions of SNAI1 and SNAI2 in corneal epithelial cells. TGF-β increases the activities of Smad, ERK, and AKT, and these molecules upregulate SNAI1 and SNAI2 expression, but not TWIST1.
Figure 7.
 
Proposed model depicting the signaling pathway of TGF-β–mediated expressions of SNAI1 and SNAI2 in corneal epithelial cells. TGF-β increases the activities of Smad, ERK, and AKT, and these molecules upregulate SNAI1 and SNAI2 expression, but not TWIST1.
Supplementary Materials
Figure sf01, PT - Figure sf01, PT 
Footnotes
 Supported by funds for the Third-Term Comprehensive 10-Year Strategy for Cancer Control, the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NiBio), and a Grant-in-Aid for Scientific Research (A).
Footnotes
 Disclosure: K. Aomatsu, None; T. Arao, None; K. Sugioka, None; K. Matsumoto, None; D. Tamura, None; K. Kudo, None; H. Kaneda, None; K. Tanaka, None; Y. Fujita, None; Y. Shimomura, None; K. Nishio, None
The authors thank Shinji Kurashimo and Yoshitaka Horiuchi for technical assistance. 
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Figure 1.
 
TGF-β signaling actually provides a signal response to human corneal epithelial cells (HCECs). (A) Western blot examining the phosphorylation and expression levels of Smad2 in time- (0, 15, 30, and 60 min of TGF-β stimulation) and dose- (0.1, 1, and 10 ng/mL) dependent responses to TGF-β stimuli in HCECs. β-actin was used as an internal control. Marker, protein size marker. (B) Growth inhibitory effect of TGF-β in HCECs evaluated using an MTT assay after incubation for 0, 24, 48, and 72 h. The experiments were performed in triplicate. The error bars represent the SD. *P < 0.05.
Figure 1.
 
TGF-β signaling actually provides a signal response to human corneal epithelial cells (HCECs). (A) Western blot examining the phosphorylation and expression levels of Smad2 in time- (0, 15, 30, and 60 min of TGF-β stimulation) and dose- (0.1, 1, and 10 ng/mL) dependent responses to TGF-β stimuli in HCECs. β-actin was used as an internal control. Marker, protein size marker. (B) Growth inhibitory effect of TGF-β in HCECs evaluated using an MTT assay after incubation for 0, 24, 48, and 72 h. The experiments were performed in triplicate. The error bars represent the SD. *P < 0.05.
Figure 2.
 
TGF-β upregulates EMT-related molecules via Smad and non-Smad pathways in HCECs. (A) The mRNA expression levels of SNAI1, SNAI2, TWIST1, VIM (vimentin), and FN1 (fibronectin 1) were determined using a real-time RT-PCR analysis. HCECs were stimulated with TGF-β at 0.1, 1, and 10 ng/mL for 24 h and the cells were used for analysis. (B) Real-time RT-PCR analysis for mRNA expression levels of SNAI1 and SNAI2 via Smad or non-Smad signaling. HCECs were cultured with or without 10 μM of the MEK inhibitor U0126, 1 μM of the phosphoinositide-3-kinase inhibitor Wortmannin, or 10 μM of the TGF-β receptor inhibitor SB431542 for 30 min and were stimulated with 10 ng/mL of TGF-β for 8 h; the cells were then collected for analysis. Rel mRNA, normalized mRNA expression levels (target gene/GAPD × 103). The experiments were performed in triplicate. The error bars represent the SD. *P < 0.05.
Figure 2.
 
TGF-β upregulates EMT-related molecules via Smad and non-Smad pathways in HCECs. (A) The mRNA expression levels of SNAI1, SNAI2, TWIST1, VIM (vimentin), and FN1 (fibronectin 1) were determined using a real-time RT-PCR analysis. HCECs were stimulated with TGF-β at 0.1, 1, and 10 ng/mL for 24 h and the cells were used for analysis. (B) Real-time RT-PCR analysis for mRNA expression levels of SNAI1 and SNAI2 via Smad or non-Smad signaling. HCECs were cultured with or without 10 μM of the MEK inhibitor U0126, 1 μM of the phosphoinositide-3-kinase inhibitor Wortmannin, or 10 μM of the TGF-β receptor inhibitor SB431542 for 30 min and were stimulated with 10 ng/mL of TGF-β for 8 h; the cells were then collected for analysis. Rel mRNA, normalized mRNA expression levels (target gene/GAPD × 103). The experiments were performed in triplicate. The error bars represent the SD. *P < 0.05.
Figure 3.
 
Protein expression regulation of N-cadherin and E-cadherin by TGF-β. (A) Western blotting for N-cadherin and E-cadherin were examined in HCECs and the EMT-positive control A549, a lung cancer cell line. HCECs were stimulated with the indicated concentration of TGF-β and the cells were collected at 24 h and 48 h. β-Actin was used as an internal control. Marker, protein size marker. (B) Western blotting for N-cadherin and E-cadherin were examined in HCECs for long time exposure. HCECs were stimulated with TGF-β at 10 ng/mL without serum starvation at indicated duration (4–6 days). β-actin was used as an internal control. (C) Immunofluorescent staining of HCECs treated with TGF-β (10 ng/mL) or without (Cont.) for 6 days. HCECs were stained for N-cadherin (upper panels) and E-cadherin (lower panels) and the nuclei of cells were counterstained with DAPI. Scale bar, 25 μm.
Figure 3.
 
Protein expression regulation of N-cadherin and E-cadherin by TGF-β. (A) Western blotting for N-cadherin and E-cadherin were examined in HCECs and the EMT-positive control A549, a lung cancer cell line. HCECs were stimulated with the indicated concentration of TGF-β and the cells were collected at 24 h and 48 h. β-Actin was used as an internal control. Marker, protein size marker. (B) Western blotting for N-cadherin and E-cadherin were examined in HCECs for long time exposure. HCECs were stimulated with TGF-β at 10 ng/mL without serum starvation at indicated duration (4–6 days). β-actin was used as an internal control. (C) Immunofluorescent staining of HCECs treated with TGF-β (10 ng/mL) or without (Cont.) for 6 days. HCECs were stained for N-cadherin (upper panels) and E-cadherin (lower panels) and the nuclei of cells were counterstained with DAPI. Scale bar, 25 μm.
Figure 4.
 
TGF-β, but not EGF, mediates EMT-like morphologic changes in HCECs. (A) Morphologic changes were evaluated using phase-contrast microscopy (upper panels) and phalloidin and DAPI staining under fluorescence microscopy (middle and lower panels). HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for 72 h and then evaluated. Scale bar: (middle panels) 100 μm; (lower panels) 25 μm. (B) The ratio of spindle-shaped cells was then analyzed (ratio of cells with twofold > length/width in a high power field). Five random fields per sample at a magnification of ×200 were captured and evaluated. The error bars represent the SD. *P < 0.05.
Figure 4.
 
TGF-β, but not EGF, mediates EMT-like morphologic changes in HCECs. (A) Morphologic changes were evaluated using phase-contrast microscopy (upper panels) and phalloidin and DAPI staining under fluorescence microscopy (middle and lower panels). HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for 72 h and then evaluated. Scale bar: (middle panels) 100 μm; (lower panels) 25 μm. (B) The ratio of spindle-shaped cells was then analyzed (ratio of cells with twofold > length/width in a high power field). Five random fields per sample at a magnification of ×200 were captured and evaluated. The error bars represent the SD. *P < 0.05.
Figure 5.
 
Inactive crosstalk between TGF-β and EGF signals in HCECs. Western blot for phospho-Smad2 and Smad2 expression (A, C) and phospho-EGFR, -ERK, and -AKT and these protein expression levels (B, D) are shown. HCECs were cultured with or without exposure to SB431542 or the EGFR tyrosine kinase inhibitor AG1478 for 30 min and stimulated with 10 ng/mL of TGF-β or EGF for 1 h or 15 min, respectively (A, B). For simultaneous stimulation with TGF-β and EGF, the HCECs were cultured with or without 10 μM of U0126, 1 μM of Wortmannin, or 1 μM of AG1478 for 30 min and were then stimulated with 10 ng/mL of TGF-β and EGF for 1 h or 15 min, respectively (C, D). β-Actin was used as an internal control. The experiments were performed in duplicate. Marker, protein size marker.
Figure 5.
 
Inactive crosstalk between TGF-β and EGF signals in HCECs. Western blot for phospho-Smad2 and Smad2 expression (A, C) and phospho-EGFR, -ERK, and -AKT and these protein expression levels (B, D) are shown. HCECs were cultured with or without exposure to SB431542 or the EGFR tyrosine kinase inhibitor AG1478 for 30 min and stimulated with 10 ng/mL of TGF-β or EGF for 1 h or 15 min, respectively (A, B). For simultaneous stimulation with TGF-β and EGF, the HCECs were cultured with or without 10 μM of U0126, 1 μM of Wortmannin, or 1 μM of AG1478 for 30 min and were then stimulated with 10 ng/mL of TGF-β and EGF for 1 h or 15 min, respectively (C, D). β-Actin was used as an internal control. The experiments were performed in duplicate. Marker, protein size marker.
Figure 6.
 
TGF-β, but not EGF, induced the sustained upregulation of SNAI1 and SNAI2. (A) The mRNA expression levels of SNAI1 and SNAI2 were determined using a real-time RT-PCR analysis. HCECs were stimulated with 10 ng/mL of TGF-β or EGF for 2 h and the cells were analyzed. (B) Detailed time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-β or EGF. HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for the indicated time course. Note that TGF-β resulted in the sustained upregulation of SNAI1 and SNAI2 for >48 h. Rel mRNA, normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. (C) Further time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-β. HCECs were cultured in the presence of 10 ng/mL of TGF-β for the indicated time course. Normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. *P < 0.05. (D) Western blotting for SNAI1/Snail1. HCECs were cultured in the presence of 10 ng/mL of TGF-β for the indicated time course and exposed to 10 μM of SB431542 at 8 h. β-actin was used as an internal control. M, protein size marker. (E) Long-term expression of SNAI1 by TGF-β or EGF. HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for the indicated time course. β-actin was used as an internal control.
Figure 6.
 
TGF-β, but not EGF, induced the sustained upregulation of SNAI1 and SNAI2. (A) The mRNA expression levels of SNAI1 and SNAI2 were determined using a real-time RT-PCR analysis. HCECs were stimulated with 10 ng/mL of TGF-β or EGF for 2 h and the cells were analyzed. (B) Detailed time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-β or EGF. HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for the indicated time course. Note that TGF-β resulted in the sustained upregulation of SNAI1 and SNAI2 for >48 h. Rel mRNA, normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. (C) Further time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-β. HCECs were cultured in the presence of 10 ng/mL of TGF-β for the indicated time course. Normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. *P < 0.05. (D) Western blotting for SNAI1/Snail1. HCECs were cultured in the presence of 10 ng/mL of TGF-β for the indicated time course and exposed to 10 μM of SB431542 at 8 h. β-actin was used as an internal control. M, protein size marker. (E) Long-term expression of SNAI1 by TGF-β or EGF. HCECs were cultured in the presence of 10 ng/mL of TGF-β or EGF for the indicated time course. β-actin was used as an internal control.
Figure 7.
 
Proposed model depicting the signaling pathway of TGF-β–mediated expressions of SNAI1 and SNAI2 in corneal epithelial cells. TGF-β increases the activities of Smad, ERK, and AKT, and these molecules upregulate SNAI1 and SNAI2 expression, but not TWIST1.
Figure 7.
 
Proposed model depicting the signaling pathway of TGF-β–mediated expressions of SNAI1 and SNAI2 in corneal epithelial cells. TGF-β increases the activities of Smad, ERK, and AKT, and these molecules upregulate SNAI1 and SNAI2 expression, but not TWIST1.
Figure sf01, PT
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