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Electric field-induced suppression of PTEN drives epithelial-to-mesenchymal transition via mTORC1 activation
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Original article
Electric field-induced suppression of PTEN drives epithelial-to -mesenchymal transition via mTORC1 activation Tiantian Yana , Xupin Jianga , Xiaowei Guob , Wen Chenc, Di Tanga , Junhui Zhanga , Xingyue Zhanga , Dongxia Zhanga , Qiong Zhanga , Jiezhi Jiaa , Yuesheng Huanga,* a Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing 400038, China b Department of Burns and Plastic Surgery, The 205th Hospital of People's Liberation Army, Jinzhou 121001, China c Department of Endocrinology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 August 2016 Received in revised form 3 November 2016 Accepted 17 November 2016
Background: Naturally occurring electric fields (EFs) are an intrinsic property of wounds. Endogenous EFs in skin wounds play critical roles in the dynamic and well-ordered biological process of wound healing. The epithelial-to-mesenchymal transition (EMT) allows keratinocytes to transition from sedentary cells to motile cells, facilitating wound healing. However, EMT-related studies have been performed without considering endogenous EFs. Thus, the relationship between electrical signals and the EMT remain elusive. Objective: Phosphatase and tension homolog (PTEN) and mammalian target of rapamycin complex 1 (mTORC1) are key molecules in sensing electrical cues, and they play significant roles in cellular responses to EFs. In addition, these molecules are closely related to the occurrence of the EMT in other cells. We used primary human keratinocytes to investigate the influence of EFs on the EMT as well as the roles of PTEN and mTORC1 in this process. Methods: The effects of EFs on the EMT were investigated by analyzing the levels of specific proteins and transcription factors. The roles of mTORC1 and PTEN and their relationship with each other were studied via pharmacological inhibition or genetic knockdown. A Zeiss imaging system and scratch assays were used to study single-cell motility and monolayer cell migration. Results: EFs induced a range of both biochemical changes (e.g., increased Snail, Slug, vimentin, and Ncadherin expression, decreased E-cadherin expression) and functional changes (e.g., enhanced migratory capacity) that are characteristic of the EMT. EF-stimulated cells exhibited suppressed PTEN expression, and further PTEN downregulation led to the acquisition of more mesenchymal features and the loss of epithelial characteristics, which was accompanied by increased migratory capacity. PTEN overexpression reversed the EF-induced EMT and inhibited the migratory capacity of keratinocytes. EF-induced mTORC1 activation was a required component of the causal relationship between PTEN suppression and the EMT, as mTORC1 inhibition reversed the EMT induced by PTEN downregulation. Conclusions: Our data demonstrate that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation, thereby revealing a new and promising role of EFs in facilitating wound reepithelialization. These results provide a novel perspective regarding the significance of EFs in wound healing; therefore, electrical stimulation offers a new avenue of wound management for improved and accelerated wound healing. © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Electric fields Epithelial-to-mesenchymal transition Keratinocyte Migration PTEN mTORC1
1. Introduction Skin fulfills multiple key functions and is the largest organ of the body. Disruption in the skin instantaneously generates
* Corresponding author. E-mail address: [email protected] (Y. Huang).
endogenous electric fields (EFs) resulting from the collapse of transepithelial potentials (TEPs); the wound center acts as the cathode of the endogenous EFs until wound reepithelialization is completed [1–3]. Endogenous EFs have been considered an overriding directional cue guiding the lateral migration direction of keratinocytes for reepithelialization [2], indicating that EFs play a critical role in the dynamic and well-ordered biological process of
http://dx.doi.org/10.1016/j.jdermsci.2016.11.007 0923-1811/ © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: T. Yan, et al., Electric field-induced suppression of PTEN drives epithelial-to-mesenchymal transition via mTORC1 activation, J Dermatol Sci (2016), http://dx.doi.org/10.1016/j.jdermsci.2016.11.007
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wound healing. The strength of endogenous EFs measured experimentally in humans and animals ranges from 42 to 100 mV/mm [1,4,5]. In vitro, the responses of cells to applied EFs comparable to the strength of endogenous EFs have confirmed the importance of EFs in many cellular physiological processes [3]. These studies have also deciphered components of the underlying mechanisms, including phosphoinositide 3-kinase (PI3 K)/phosphatase and tension homolog (PTEN), integrins and membrane growth factor receptors [6]. Thus, endogenous EFs are vital and significant occurrences that should be taken into consideration in studies related to wound healing. The essence of wound healing is the restoration of the epidermal barrier against the external environment [7]. Keratinocyte-driven reepithelialization is achieved through reduced intercellular adhesion and enhanced migration in the epidermis proximal to the wound margin and directly contributes to wound closure [8–11]. The initial step, i.e., the acquisition of migratory capability, is an essential part of this cascade [12]. The term “epithelial-to-mesenchymal transition” (EMT) refers to an intricate process that allows keratinocytes to transition from being sedentary to motile through changes in cellular physiology and molecular biology. During the EMT, differentiated, polarized keratinocytes undergo reprogramming and acquire mesenchymal features, including a lack of apical–basal polarity, a loss of cell–cell adhesion, and enhanced motility [7,12]. Many molecular processes are engaged in order to initiate and complete the EMT, including the activation of transcription factors, the reorganization and expression of cytoskeletal proteins, and changes in the expression of specific proteins. These involved factors are also used as basic biomarkers to demonstrate the passage of a cell through the EMT [13,14]. The EMT is closely related to wound healing and is orchestrated by injury signals, such as tumor necrosis factor a and transforming growth factor b. Although the underlying mechanisms of the EMT occurring in keratinocytes have been thoroughly discussed [7,8,15–19], the relevant experiments were performed under conventional culture conditions, ignoring the endogenous EFs that keratinocytes initially experience even prior to their migration in vivo [3]. Therefore, it is necessary to elucidate the regulatory mechanisms of EFs in the EMT during wound healing. PTEN encodes a lipid and protein phosphatase and has been identified as a tumor suppressor that is negatively correlated with cell migration. PTEN plays a pivotal role in regulating tumor growth, migration, and metastasis; additionally, its mutation or deletion is frequently found in different cancers and is associated with the EMT [20]. Coincidentally, PTEN expression is decreased significantly in various cells subjected to EFs applied in vitro [21,22], and its down-regulation or deletion can enhance EFdirected keratinocyte migration [1]. However, whether PTEN contributes to the EMT in keratinocytes exposed to EFs has yet to be determined. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved kinase that serves as an integrator of extracellular information [23]. mTOR exists in two complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 consists of mTOR, Raptor, and mLST8, among other components. The critical role of mTORC1 in the EMT has been demonstrated via genetic modification and pharmacological regulation; and its up- regulation or down-regulation is closely related to the gain or loss characteristic features of the EMT, respectively. Additionally, numerous studies have revealed the causal relationship between PTEN and mTORC1. These reports and observed results raised the hypothesis that PTEN and mTORC1 may be involved in the possible relationship between EFs and the EMT. To investigate these possibilities, we examined the occurrence of the EMT in keratinocytes exposed to applied EFs by assessing well-established biomarkers and cell motility. The EMT characteristics were closely associated with the expression of PTEN and
mTORC1, indicating a possible causative mechanism and prompting further investigation into the roles that PTEN and mTORC1 play in the EMT. We found that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation. 2. Materials and methods 2.1. Cell lines, reagents and siRNA transfections Primary human keratinocytes (HKs) were kind gifts from Song Wang. In brief, the foreskin was treated with 0.25% trypsin/0.04% EDTA solution (Invitrogen, USA) at 4 C for 24 h and then seeded into dishes pre-coated with type IV collagen. HKs were cultured in keratinocyte serum-free medium (Gibco, USA) and all cells were routinely cultured at 37 C in a 5% CO2 atmosphere. Medium with or without Rapamycin (100 mM, Sigma) was added, and the cells were pre-incubated at 37 C for 4 h. The cells were then washed, and the medium was refreshed. siRNA directed against PTEN was obtained from Shanghai GeneChem, Co., Ltd. (Shanghai, China), and siNC, the non-targeting control sequence, was used to create control cells. All the transfections were performed in accordance with the manufacturer’s instructions. 2.2. EF stimulation and imaging of single-cell motility The strength of the applied EFs (100 mV/mm) was based both on the related literature and our previous study. EF stimulation was utilized as previously described [3]. In brief, keratinocytes were stimulated by EFs applied via two silver electrodes immersed in Steinberg’s solution, which was connected to the culture medium by two agar bridges. After cells were exposed to EFs, time-lapse imaging was performed using a Zeiss imaging system (Carl Zeiss Meditec, Jena, Germany) to monitor the motility of single cells visually unaffected by proliferation or death. Images were analyzed using NIH ImageJ software (http://rsb.info.nih.gov/ij/). The velocity of cells was calculated as their movement trajectories divided by time (min), reflecting single-cell motility. 2.3. Wound-healing assays Scratch assays were used to determine the migratory response of monolayered cells. Cells were grown to full confluence, stimulated by EFs and scratched with a pipette tip to create a cell-free gap. The cells were then washed with medium to remove cell debris and were cultured for 12 h. Images were captured every 6 h using an inverted phase-contrast microscope. The percent of wound closure in three randomly chosen fields was calculated with NIH Image J software. 2.4. Western blot Protein extracts were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and blocked with 5% non-fat milk or bovine serum albumin. The desired bands were incubated with the appropriate primary and secondary antibodies and were subsequently visualized using chemiluminescence reagents (Pierce, Rockford, IL). Antibodies for PTEN, phospho-p70S6 kinase (Thr389), p70S6K, phospho-4E-BP1 (Thr70), 4E-BP1, vimentin, Ecadherin, N-cadherin, Snail, Slug, b-actin, and HRP-linked secondary antibody were obtained from Cell Signaling (Danvers, MA). 2.5. Immunofluorescence (IF) staining Cells stimulated by EFs were fixed with 4% paraformaldehyde and blocked with 3% goat serum albumin to diminish background signals. The cells were subsequently incubated with the primary
Please cite this article in press as: T. Yan, et al., Electric field-induced suppression of PTEN drives epithelial-to-mesenchymal transition via mTORC1 activation, J Dermatol Sci (2016), http://dx.doi.org/10.1016/j.jdermsci.2016.11.007
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antibody, corresponding fluorescence-labeled secondary antibody, and DAPI. The slides were imaged using a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany). 2.6. Statistical analysis All experiments were performed in duplicate and repeated at least three times. The data are presented as the mean standard deviation (SD). Statistical significance among multiple groups was evaluated using a Student’s t-test. P values < 0.05 were considered to be statistically significant. 3. Results 3.1. EFs induce the EMT with enhanced motility and migration The molecular processes involved in the EMT were utilized as biomarkers to demonstrate the occurrence of the EMT in cells. The activation of transcription factors (Snail and Slug), the increased expression of cytoskeletal marker (vimentin), and the switch from E-cadherin to N-cadherin are all hallmarks of the EMT and are accompanied by increased migratory capacity. Therefore, timecourse experiments were performed, in which HKs were stimulated by EFs for different durations. As shown in Fig. 1A, the EFs increased the expression of Snail and Slug in a time-dependent manner, and this increase was initially detectable 2 h after the application of EFs. Along with the increase in vimentin and the cadherin switch, these changes suggest that treatment with EFs causes the loss of epithelial characteristics and the acquisition of mesenchymal features. To further study specific expression changes associated with the EMT, we immunofluorescently stained EF-stimulated cells (6 h) for vimentin and E-cadherin expression, which are frequently used to monitor the progression of the EMT. Representative samples for each protein are shown in Fig. 1B. E-cadherin was positively expressed in almost all the cells but was suppressed after the EF treatment. Normal cells demonstrated either negative or mild staining for vimentin; meanwhile, EF-stimulated cells exhibited wide cytoplasmic staining for vimentin. These changes are in complete agreement with the corresponding Western blot results. Enhanced migratory capacity is a critical feature of the EMT that facilitates reepithelialization. Images recording cell trajectories showed that EF stimulation greatly increased their range of movement. The statistical analysis of the velocity confirmed the positive effect of the EF treatment on the EMT via increased motility (Fig. 1C). The effect of EFs on migratory capacity was further confirmed using scratch assays. As shown by the representatively results in Fig. 1D, cells stimulated by EFs migrated into nearly 67% of the original cell-free area while the area covered by normal cells was only 33% after 12 h. The statistical analysis of the monolayer migration assays demonstrated a positive correlation between the EF treatment and the enhanced migratory capacity, which was consistent with the single-cell motility results (P < 0.05). Taken together, these findings demonstrate that EFs equivalent in strength to physiological EFs found in vivo stimulate keratinocyte mobilization and gain of mesenchymal features. 3.2. EF-induced suppression of PTEN drives the EMT acquisition After verifying the occurrence of the EF-induced EMT, we speculated that there might be a signal relaying electrical cues into cellular responses. Notably, PTEN plays a critical role in these two processes, which prompted us to study the possibility that PTEN suppression is correlated with the EF-induced EMT. After demonstrating that no differences existed between normal cells and siNC-transfected cells (data not shown), we investigated
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whether EFs induce PTEN down regulation. As clearly presented in Fig. 2A, EFs suppressed the expression of PTEN in a timedependent manner. Then, the role of PTEN in the EF-induced EMT was evaluated by characterizing the effects of further downregulating PTEN expression using siPTEN and over-expressing PTEN expression using recombinant adenovirus vectors (AdPTEN). No changes in transcription factors or protein markers were observed in cells transfected with siNC and Vector. Meanwhile, cells treated with EFs exhibited EMT, with the loss-of-epithelial markers and gain-of-mesenchymal features. Further-decreased PTEN expression dramatically promoted the EF-induced EMT, as illustrated by the increased expression of Snail, Slug, vimentin and N-cadherin accompanied by the decreased E-cadherin expression (Fig. 2B). Similar to the Western blot results, the IF findings also showed that PTEN knockdown increased vimentin expression and decreased E-cadherin expression (Fig. 2C). As shown by the Western blot, PTEN overexpression inhibited EMT acquisition (Supplementary Fig. S1A). Additionally, IF results showed that PTEN overexpression under normal culture conditions induced higher E-cadherin expression (stronger relative fluorescence intensity, data not shown) and almost entirely abolished vimentin expression (lower relative fluorescence intensity, data not shown). PTEN overexpression in EFs-stimulated cells also increased Ecadherin expression and decreased vimentin expression, reflecting inhibition of EMT (Supplementary Fig. S1B). To determine whether PTEN is a mediator of EMT-associated migration, we measured the movement ranges and coverage areas in the cell motility and monolayer migration assays, respectively. As expected, the knockdown of PTEN further increased the movement range of cells compared with that of the siNCtransfected control cells (Fig. 2D). The siNC-transfected cells that had undergone the EMT after the EF pretreatment achieved 76% wound closure, and silencing PTEN expression resulted in nearly 95% coverage of the original wound (Fig. 2E). In addition, PTEN overexpression inhibited the single cell motility and monolayer migration of keratinocytes (Supplementary Fig. S1C and D). These results define PTEN as a regulator of the EMT in keratinocytes when exposed to EFs; how PTEN regulates the EMT requires further study. 3.3. mTORC1 is required for the EF-induced EMT and enhanced migratory capacity mTORC1 attracted our attention when studying how PTEN regulates the EMT since it is coupled with PTEN in underlying the cellular responses to EFs. After demonstrating that no differences existed between normal cells and cells treated with DMSO (data not shown), we first determined whether mTORC1 is involved in the EFs-induced responses and EMT. Phospho-p70S6 kinase (Thr389) and phospho-4E-BP1 (Thr70) serve as indicators of mTORC1 activity, which can be visualized using commercially available antibodies. In the time-course experiments, the levels of p70S6K and 4E-BP1 phosphorylation were dramatically increased with prolonged stimulation times. Total p70S6K and 4E-BP1 levels were not affected by the EF treatment (Fig. 3A). Rapamycin is a classical inhibitor of mTORC1 and was used to decipher the role mTORC1 plays in regulating the EMT. Cells incubated with Rapamycin demonstrated over a 90% reduction in phospho-p70S6K and phospho-4E-BP1 levels. Since the targeted inhibition of mTORC1 was confirmed, we explored whether its inhibition is sufficient to attenuate the EF-induced EMT by examining the expression levels of the aforementioned markers. As shown in Fig. 3B, the EF-induced induction of Snail, Slug, vimentin, and N-cadherin expression and decrease in E-cadherin expression were attenuated in keratinocytes with inhibited mTORC1. IF confirmed the increased levels of E-cadherin (stronger
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Fig. 1. EFs induce the EMT with enhanced motility and migration. (A) Western blot results of cells stimulated by EFs and probed for Snail, Slug, vimentin, E-cadherin, Ncadherin, b-actin (loading control). (B) IF staining for E-cadherin (green), vimentin (red) and the nuclear compartment (blue) in control and EF-stimulated cells. Scale bar = 25 mm. (C) The movement trajectories of cells in the control and EF-stimulated groups were recorded by a Zeiss imaging system. The velocity of each group is shown as the mean SD. *P < 0.05 versus the control group. (D) Scratch assays were used to confirm the migration-promoting effect of EFs in keratinocytes. The wound closure (%) was calculated by the reduction of the wound area over time and is shown as the mean SD. *P < 0.05 versus the control group. Scalebar = 200 mm.
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Fig. 2. EF-induced suppression of PTEN contributes to both the EMT and migratory capacity. (A) Immunoblot analysis for PTEN expression in control and EF-stimulated cells. (B) Immunoblot for the expression of PTEN and EMT markers in siRNA-transfected cells with or without EF treatment. (C) IF staining in control (transfected with siNC), siPTEN, 6-h EF-treated (transfected with siNC or siPTEN) cells. Scale bar = 25 mm. (D) The movement trajectories of control (transfected with siNC), siPTEN, 6-h EF-treated (transfected with siNC or siPTEN) cells. *P < 0.05 versus the control group. #P < 0.05 versus the 6-h group. (E) Monolayer migration in control (transfected with siNC), siPTEN, 6-h EFtreated (transfected with siNC or siPTEN) cells. *P < 0.05 versus the control group. #P < 0.05 versus the 6-h group. Scalebar = 200 mm.
relative fluorescence intensity, data not shown) and the decreased levels (nearly abolished) of vimentin (lower relative fluorescence intensity, data not shown) in mTORC1 inhibited groups (Rapamycin, 6h + Rapamycin) compared with the N group and 6-h group, respectively (Fig. 3 C). The migratory capacity of the cells above was assessed using a Zeiss imaging system and scratch assays. mTORC1 inhibition
significantly decreased the movement range of cells and velocity of the keratinocytes (Fig. 3D). Consistent with these results, mTORC1 inhibition significantly decreased the migration of cells exposed to EFs, as demonstrated using a wound-healing assay (Fig. 3E). Collectively, these data show that mTORC1 is both activated in EFtreated keratinocytes and required for the EMT, an important step in the initiation of reepithelialization during wound healing.
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Fig. 3. mTORC1 is required for EF-induced EMT and enhanced migratory capacity. (A) Western blot detecting the activity of mTORC1 in EF-treated cells through the expression levels of phosphor-p70S6K (Thr389), p70S6K, phosphor-4E-BP1 (Thr70), 4E-BP1, and b-actin (loading control). (B) Western blot demonstrating changes in mTORC1 activity and EMT markers levels in control and EF-stimulated cells treated with DMSO or Rapamycin. (C) IF staining for E-cadherin (green), vimentin (red) and the nuclear compartment (blue) in control cells (treated with DMSO or Rapamycin) and 6-h EF-stimulated cells treated with DMSO or Rapamycin. Scale bar = 25 mm. (D) The movement trajectories of control cells (treated with DMSO or Rapamycin) and 6-h EF-stimulated cells treated with DMSO or Rapamycin. *P < 0.05 versus the control group. #P < 0.05
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3.4. mTORC1 is involved in the occurrence of the EMT driven by PTEN suppression Our results show that PTEN suppression and mTORC1 activation are both required for gaining mesenchymal features and enhanced migratory capacity, which normally accompany the EMT. Considering the reports in the literatures demonstrating that PTEN can suppress mTORC1, we reasoned that during the EMT process, there might be a causal relationship between PTEN and mTORC1. To test this possibility, we first showed that no differences existed among normal cells and cells treated with DMSO, siNC or both (data not shown); then, we detected the activity of mTORC1 in cells after PTEN-knockdown and found that the levels of p70S6K and 4E-BP1 phosphorylation were further increased compared with those of cells exposed to EFs, verifying that PTEN can regulate mTORC1 activity. Meanwhile, the expression level of PTEN was not affected by mTORC1 inhibition. Consistently, the changes of the aforementioned EMT markers demonstrated that PTEN knockdown promoted the occurrence of the EMT in EF-stimulated cells. This PTEN suppression-related EMT was attenuated in cells with inhibited mTORC1, as shown in Fig. 4A, suggesting that mTORC1 is necessary for the occurrence of the EMT induced by PTEN suppression (Fig. 4A). Coincidentally, similar results were found by IF. EFstimulated cells showed downregulated E-cadherin compared to normal cells (data not shown), and siPTEN further reduced the expression of E-cadherin, which could be reversed by Rapamycin treatment. Similar results were also found via vimentin staining (Fig. 4B). We utilized a Zeiss imaging system and scratch assays to determine whether mTORC1 is essential for the enhanced migratory capacity of PTEN-knockdown cells. Cells treated with EFs had larger movement ranges than normal cells (data not shown), which was consistent with the results described above. PTEN knockdown further enlarged the movement range of cells treated with EFs. With the inhibition of both PTEN and mTORC1, the trajectories of cells were substantially decreased compared with the cells that were only targeted for PTEN knockdown (Fig. 4C). Cells that were stimulated by EFs and underwent the EMT achieved 70% wound closure. Meanwhile, the targeted knockdown of only PTEN resulted in nearly 95% coverage of the original cellfree area, while the additional inhibition of mTORC1 reversed the PTEN downregulation-induced migration, leading to only 40% wound coverage (Fig. 4D). Collectively, our results acquired by integrating multiple experimental approaches demonstrate that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation. 4. Discussion Upon injury, skin disruption instantaneously generates EFs due to the collapse of TEPs. Accumulating evidence indicates that EFs are vital and significant occurrences to which keratinocytes are subjected. To repair skin wounds effectively, keratinocytes from the wound margin undergo the EMT, which allows them to lose their epithelial characteristics and acquire mesenchymal features and migratory properties. However, related studies that have not considered these EFs may ultimately be unable to reflect the complexity of the EMT of keratinocytes as they respond to changing environments[24]. In this study, we determined the role of EFs in regulating the EMT in keratinocytes for the first time. First, we demonstrated that EFs induce the EMT along with enhanced migratory abilities, which normally accompany the EMT (Fig. 1).
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Second, we found that the EF-induced inhibition of PTEN and activation of mTORC1 are both greatly involved in the EMT (Figs. 2 and 3, Supplementary Fig. S1). These findings identify PTEN and mTORC1 as functional molecules in the EF-induced EMT and important regulators of EMT processes. Third, we showed that the inhibition of mTORC1 reversed the PTEN suppression-induced EMT and migratory capacity concomitant with attenuated EMT hallmarks (Fig. 4), suggesting that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation (Fig. 5). The EMT is a key reversible step that facilitates reepithelialization as well as tumor metastasis and invasion. Due to the clinical significance of this process, the induction of the EMT is an appealing therapeutic approach that could meaningfully expedite wound repair. Our findings implicate EFs as key regulators of the EMT in keratinocytes, which is achieved by PTEN modulation and mTORC1 activation. This conclusion is grounded on the observations that applied EFs induced a range of biochemical changes (e.g., increased expression of Snail, Slug, vimentin, N-cadherin, and decreased E-cadherin expression) and functional chang es (e.g., enhanced migratory capacity) that are characteristic of the EMT [12]. This new discovery further highlights the importance of EFs in wound healing, as the EMT enables keratinocytes to transform from sedentary cells into motile cells. In this context, we suggest a novel role for EFs in the support of wound repair by inducing the EMT. In normal skin, keratinocytes go through a process of terminal differentiation and provide a barrier that protects the underlying tissue from pathogenic microorganisms invasion and mechanical, chemical, and physical damage. The barrier function is enhanced by cell–substrate and cell–cell interactions, such as tight junctions and adherens junctions [25–28]. Upon wounding, the cells from the wound margin proceed through the EMT and transform from proliferative, sedentary cells into migratory cells; this process is essential for successfully achieving reepithelialization. During the EMT, Snail (also known as Snail1) and Slug (also known as Snail2), as transcription factors, repress the expression of cell junction proteins [29–31]. The downregulation of E-cadherin is balanced by the increased level of N-cadherin. Through this cadherin switch, the transitioning cells acquire N-cadherin interactions, which are weaker than E-cadherin interactions and facilitate cell motility and migration. Additionally, the induced expression of vimentin is closely related with the other EMT hallmarks and regulates both the membrane-associated protein activity and organelles trafficking. These changes are widely accepted biomarkers of the EMT. In our study, the fact that EFs were found to induce the EMT by studying the aforementioned markers raises a legitimate question: how are electrical cues relayed into cellular responses causing the EMT? PI3K and PTEN are key molecules in sensing electrical cues. EFs activate PI3K signaling in keratinocytes rapidly and specifically [9]. PTEN is a negative regulator of PI3K and has been shown to be downregulated in several cell types [22]. Therefore, its deletion enhances PI3K activation and cellular reactivity to EFs [1]. Thus, PI3K and PTEN represent molecules and genes underlying cellular responses to EFs. The mTOR signaling pathway is an important component of PI3K signaling, and it integrates multiple types of information, such as the levels of energy, growth factors and hypoxia, to facilitate the migration and invasion of cancer cells. While intensive studies have focused on the role of mTORC1 in regulating the EMT, growth, survival, and motility of cancer cells under normal conditions, its role in EF-treated keratinocytes and the related cellular responses has not been extensively studied. We found that the pharmacological inhibition of mTORC1 significantly
versus the 6-h group. (E) Monolayer migration in control cells (treated with DMSO or Rapamycin) and 6-h EF-stimulated cells treated with DMSO or Rapamycin. *P < 0.05 versus the control group. #P < 0.05 versus the 6-h group. Scalebar = 200 mm.
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Fig. 4. mTORC1 is involved in the acquisition of EMT driven by PTEN suppression. (A) Western blot results for cells treated with siNC, siPTEN, DMSO or Rapamycin detecting the expression levels of PTEN, phosphor-p70S6K (Thr389), p70S6 K, phosphor-4E-BP1 (Thr70), 4E-BP1 and EMT markers. (B) IF staining for vimentin, E-cadherin and the nuclear compartment in groups treated as described in (A). Scale bar = 25 mm. (C) The movement trajectories of groups treated as described in (A) were recorded and analyzed. *P < 0.05 versus the 6-h group. #P < 0.05 versus the siPTEN group. (D) The scratch assay results, represented as wound closure, of the group treated as described in (A) were recorded and analyzed and are shown as the mean SD. *P < 0.05 versus the 6-h group. #P < 0.05 versus the siPTEN group. Scalebar = 200 mm.
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Fig. 5. Schematic illustrating that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation during wound healing.
reversed EF-induced and PTEN-knockdown-related EMT, which was accompanied by attenuated migratory ability. Our results are consistent with those of previous studies demonstrating that the inhibition of mTORC1 in various cancer types potently inhibited the EMT and motility induced by TGFb, IGF-1 and EGF [12,32–34], suggesting that multiple signals ultimately converge upon mTORC1. As a negative regulator, PTEN deletion or knockdown has also been demonstrated to activate mTORC1 and facilitate improved motility and EMT acquisition. These discoveries highlight the fact that these functions of PTEN and its interaction partners are not cell-type specific and occur in various types of circumstances and cells. However, our study is just a preliminary exploration of the potential signal network underlying EFs and the EMT, and these findings have raised many intriguing questions. How is PTEN downregulated by EFs? Do any other molecules sense and relay electrical cues into cellular responses causing the EMT? How does mTORC1 link the expression of transcription factors and changes in typical protein markers? Is there crosstalk among other important components of the PI3K pathway, such as mTORC2? Future studies may further elucidate this powerful component of wound healing and regeneration. Endogenous EFs are an intrinsic property of wounds. Clinically, the manipulation of EFs is a new and appealing avenue for improving and accelerating wound healing. Alternatively, pharmacologically enhancing the naturally occurring EFs could serve as a potent and promising approach toward developing wound-healing therapies. For instance, ascorbic acid and AgNO3 can activate Na+ channels and increase the TEPs and EFs [35–38]. Such drugs that converge on one common denominator, i.e., altered EFs, appear to be good candidates for accelerating wound healing. In conclusion, we have demonstrated for the first time that EFinduced PTEN repression drives the EMT via mTORC1 activation. These results provide a novel perspective of the significance of EFs in wound healing, which could lead to new wound management strategies. Author contributions T.Y., Y.H., X.J. developed the initial concept. Y.H. and X.J. designed the experiments, T.Y., X.G., W.C., D.T., J.Z., X.Z., Q.Z. and J.J. performed the research and analyzed the data. T.Y., D.Z., Y.H., D.T.
wrote the manuscript; Y.H. supervised the study. All authors discussed the results and commented on the manuscript. Conflict of interests The authors have no conflicts of interest to declare. Acknowledgements This work was supported by State Key Development Program for Basic Research of China (grant number 2012CB518101) and Research fund of the State Key laboratory of Trauma, Burns and Combined injury (grant numbers SKLZZ201203; SKLZZ2012(III)01). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2016.11.007. References [1] M. Zhao, B. Song, J. Pu, T. Wada, B. Reid, G. Tai, et al., Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN, Nature 442 (2006) 457–460. [2] M. Zhao, Electrical fields in wound healing–an overriding signal that directs cell migration, Semin. Cell Dev. Biol. 20 (2009) 674–682. [3] X. Guo, X. Jiang, X. Ren, H. Sun, D.Q. Zhang, et al., The galvanotactic migration of keratinocytes is enhanced by hypoxic preconditioning, Sci. Rep. 5 (2015) 10289. [4] M. Chiang, K.R. Robinson, J.W. Vanable Jr., Electrical fields in the vicinity of epithelial wounds in the isolated bovine eye, Exp. Eye Res. 54 (1992) 999– 1003. [5] A.T. Barker, L.F. Jaffe, J.W. Vanable Jr., The glabrous epidermis of cavies contains a powerful battery, Am. J. Physiol. 242 (1982) R358–366. [6] M. Zhao, J. Penninger, R.R. Isseroff, Electrical activation of wound-healing pathways, Adv. Skin Wound Care 1 (2010) 567–573. [7] C. Yan, W.A. Grimm, W.L. Garner, L. Qin, T. Travis, N. Tan, et al., Epithelial to mesenchymal transition in human skin wound healing is induced by tumor necrosis factor-alpha through bone morphogenic protein-2, Am. J. Pathol. 176 (2010) 2247–2258. [8] M. Nakamura, Y. Tokura, Epithelial-mesenchymal transition in the skin, J. Dermatol. Sci. 61 (2011) 7–13. [9] P. Martin, Wound healing–aiming for perfect skin regeneration, Science 276 (1997) 75–81. [10] M.C. Heng, Wound healing in adult skin: aiming for perfect regeneration, Int. J. Dermatol. 50 (2011) 1058–1066.
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Original article
Electric field-induced suppression of PTEN drives epithelial-to -mesenchymal transition via mTORC1 activation Tiantian Yana , Xupin Jianga , Xiaowei Guob , Wen Chenc, Di Tanga , Junhui Zhanga , Xingyue Zhanga , Dongxia Zhanga , Qiong Zhanga , Jiezhi Jiaa , Yuesheng Huanga,* a Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing 400038, China b Department of Burns and Plastic Surgery, The 205th Hospital of People's Liberation Army, Jinzhou 121001, China c Department of Endocrinology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 August 2016 Received in revised form 3 November 2016 Accepted 17 November 2016
Background: Naturally occurring electric fields (EFs) are an intrinsic property of wounds. Endogenous EFs in skin wounds play critical roles in the dynamic and well-ordered biological process of wound healing. The epithelial-to-mesenchymal transition (EMT) allows keratinocytes to transition from sedentary cells to motile cells, facilitating wound healing. However, EMT-related studies have been performed without considering endogenous EFs. Thus, the relationship between electrical signals and the EMT remain elusive. Objective: Phosphatase and tension homolog (PTEN) and mammalian target of rapamycin complex 1 (mTORC1) are key molecules in sensing electrical cues, and they play significant roles in cellular responses to EFs. In addition, these molecules are closely related to the occurrence of the EMT in other cells. We used primary human keratinocytes to investigate the influence of EFs on the EMT as well as the roles of PTEN and mTORC1 in this process. Methods: The effects of EFs on the EMT were investigated by analyzing the levels of specific proteins and transcription factors. The roles of mTORC1 and PTEN and their relationship with each other were studied via pharmacological inhibition or genetic knockdown. A Zeiss imaging system and scratch assays were used to study single-cell motility and monolayer cell migration. Results: EFs induced a range of both biochemical changes (e.g., increased Snail, Slug, vimentin, and Ncadherin expression, decreased E-cadherin expression) and functional changes (e.g., enhanced migratory capacity) that are characteristic of the EMT. EF-stimulated cells exhibited suppressed PTEN expression, and further PTEN downregulation led to the acquisition of more mesenchymal features and the loss of epithelial characteristics, which was accompanied by increased migratory capacity. PTEN overexpression reversed the EF-induced EMT and inhibited the migratory capacity of keratinocytes. EF-induced mTORC1 activation was a required component of the causal relationship between PTEN suppression and the EMT, as mTORC1 inhibition reversed the EMT induced by PTEN downregulation. Conclusions: Our data demonstrate that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation, thereby revealing a new and promising role of EFs in facilitating wound reepithelialization. These results provide a novel perspective regarding the significance of EFs in wound healing; therefore, electrical stimulation offers a new avenue of wound management for improved and accelerated wound healing. © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Electric fields Epithelial-to-mesenchymal transition Keratinocyte Migration PTEN mTORC1
1. Introduction Skin fulfills multiple key functions and is the largest organ of the body. Disruption in the skin instantaneously generates
* Corresponding author. E-mail address: [email protected] (Y. Huang).
endogenous electric fields (EFs) resulting from the collapse of transepithelial potentials (TEPs); the wound center acts as the cathode of the endogenous EFs until wound reepithelialization is completed [1–3]. Endogenous EFs have been considered an overriding directional cue guiding the lateral migration direction of keratinocytes for reepithelialization [2], indicating that EFs play a critical role in the dynamic and well-ordered biological process of
http://dx.doi.org/10.1016/j.jdermsci.2016.11.007 0923-1811/ © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: T. Yan, et al., Electric field-induced suppression of PTEN drives epithelial-to-mesenchymal transition via mTORC1 activation, J Dermatol Sci (2016), http://dx.doi.org/10.1016/j.jdermsci.2016.11.007
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wound healing. The strength of endogenous EFs measured experimentally in humans and animals ranges from 42 to 100 mV/mm [1,4,5]. In vitro, the responses of cells to applied EFs comparable to the strength of endogenous EFs have confirmed the importance of EFs in many cellular physiological processes [3]. These studies have also deciphered components of the underlying mechanisms, including phosphoinositide 3-kinase (PI3 K)/phosphatase and tension homolog (PTEN), integrins and membrane growth factor receptors [6]. Thus, endogenous EFs are vital and significant occurrences that should be taken into consideration in studies related to wound healing. The essence of wound healing is the restoration of the epidermal barrier against the external environment [7]. Keratinocyte-driven reepithelialization is achieved through reduced intercellular adhesion and enhanced migration in the epidermis proximal to the wound margin and directly contributes to wound closure [8–11]. The initial step, i.e., the acquisition of migratory capability, is an essential part of this cascade [12]. The term “epithelial-to-mesenchymal transition” (EMT) refers to an intricate process that allows keratinocytes to transition from being sedentary to motile through changes in cellular physiology and molecular biology. During the EMT, differentiated, polarized keratinocytes undergo reprogramming and acquire mesenchymal features, including a lack of apical–basal polarity, a loss of cell–cell adhesion, and enhanced motility [7,12]. Many molecular processes are engaged in order to initiate and complete the EMT, including the activation of transcription factors, the reorganization and expression of cytoskeletal proteins, and changes in the expression of specific proteins. These involved factors are also used as basic biomarkers to demonstrate the passage of a cell through the EMT [13,14]. The EMT is closely related to wound healing and is orchestrated by injury signals, such as tumor necrosis factor a and transforming growth factor b. Although the underlying mechanisms of the EMT occurring in keratinocytes have been thoroughly discussed [7,8,15–19], the relevant experiments were performed under conventional culture conditions, ignoring the endogenous EFs that keratinocytes initially experience even prior to their migration in vivo [3]. Therefore, it is necessary to elucidate the regulatory mechanisms of EFs in the EMT during wound healing. PTEN encodes a lipid and protein phosphatase and has been identified as a tumor suppressor that is negatively correlated with cell migration. PTEN plays a pivotal role in regulating tumor growth, migration, and metastasis; additionally, its mutation or deletion is frequently found in different cancers and is associated with the EMT [20]. Coincidentally, PTEN expression is decreased significantly in various cells subjected to EFs applied in vitro [21,22], and its down-regulation or deletion can enhance EFdirected keratinocyte migration [1]. However, whether PTEN contributes to the EMT in keratinocytes exposed to EFs has yet to be determined. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved kinase that serves as an integrator of extracellular information [23]. mTOR exists in two complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 consists of mTOR, Raptor, and mLST8, among other components. The critical role of mTORC1 in the EMT has been demonstrated via genetic modification and pharmacological regulation; and its up- regulation or down-regulation is closely related to the gain or loss characteristic features of the EMT, respectively. Additionally, numerous studies have revealed the causal relationship between PTEN and mTORC1. These reports and observed results raised the hypothesis that PTEN and mTORC1 may be involved in the possible relationship between EFs and the EMT. To investigate these possibilities, we examined the occurrence of the EMT in keratinocytes exposed to applied EFs by assessing well-established biomarkers and cell motility. The EMT characteristics were closely associated with the expression of PTEN and
mTORC1, indicating a possible causative mechanism and prompting further investigation into the roles that PTEN and mTORC1 play in the EMT. We found that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation. 2. Materials and methods 2.1. Cell lines, reagents and siRNA transfections Primary human keratinocytes (HKs) were kind gifts from Song Wang. In brief, the foreskin was treated with 0.25% trypsin/0.04% EDTA solution (Invitrogen, USA) at 4 C for 24 h and then seeded into dishes pre-coated with type IV collagen. HKs were cultured in keratinocyte serum-free medium (Gibco, USA) and all cells were routinely cultured at 37 C in a 5% CO2 atmosphere. Medium with or without Rapamycin (100 mM, Sigma) was added, and the cells were pre-incubated at 37 C for 4 h. The cells were then washed, and the medium was refreshed. siRNA directed against PTEN was obtained from Shanghai GeneChem, Co., Ltd. (Shanghai, China), and siNC, the non-targeting control sequence, was used to create control cells. All the transfections were performed in accordance with the manufacturer’s instructions. 2.2. EF stimulation and imaging of single-cell motility The strength of the applied EFs (100 mV/mm) was based both on the related literature and our previous study. EF stimulation was utilized as previously described [3]. In brief, keratinocytes were stimulated by EFs applied via two silver electrodes immersed in Steinberg’s solution, which was connected to the culture medium by two agar bridges. After cells were exposed to EFs, time-lapse imaging was performed using a Zeiss imaging system (Carl Zeiss Meditec, Jena, Germany) to monitor the motility of single cells visually unaffected by proliferation or death. Images were analyzed using NIH ImageJ software (http://rsb.info.nih.gov/ij/). The velocity of cells was calculated as their movement trajectories divided by time (min), reflecting single-cell motility. 2.3. Wound-healing assays Scratch assays were used to determine the migratory response of monolayered cells. Cells were grown to full confluence, stimulated by EFs and scratched with a pipette tip to create a cell-free gap. The cells were then washed with medium to remove cell debris and were cultured for 12 h. Images were captured every 6 h using an inverted phase-contrast microscope. The percent of wound closure in three randomly chosen fields was calculated with NIH Image J software. 2.4. Western blot Protein extracts were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and blocked with 5% non-fat milk or bovine serum albumin. The desired bands were incubated with the appropriate primary and secondary antibodies and were subsequently visualized using chemiluminescence reagents (Pierce, Rockford, IL). Antibodies for PTEN, phospho-p70S6 kinase (Thr389), p70S6K, phospho-4E-BP1 (Thr70), 4E-BP1, vimentin, Ecadherin, N-cadherin, Snail, Slug, b-actin, and HRP-linked secondary antibody were obtained from Cell Signaling (Danvers, MA). 2.5. Immunofluorescence (IF) staining Cells stimulated by EFs were fixed with 4% paraformaldehyde and blocked with 3% goat serum albumin to diminish background signals. The cells were subsequently incubated with the primary
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antibody, corresponding fluorescence-labeled secondary antibody, and DAPI. The slides were imaged using a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany). 2.6. Statistical analysis All experiments were performed in duplicate and repeated at least three times. The data are presented as the mean standard deviation (SD). Statistical significance among multiple groups was evaluated using a Student’s t-test. P values < 0.05 were considered to be statistically significant. 3. Results 3.1. EFs induce the EMT with enhanced motility and migration The molecular processes involved in the EMT were utilized as biomarkers to demonstrate the occurrence of the EMT in cells. The activation of transcription factors (Snail and Slug), the increased expression of cytoskeletal marker (vimentin), and the switch from E-cadherin to N-cadherin are all hallmarks of the EMT and are accompanied by increased migratory capacity. Therefore, timecourse experiments were performed, in which HKs were stimulated by EFs for different durations. As shown in Fig. 1A, the EFs increased the expression of Snail and Slug in a time-dependent manner, and this increase was initially detectable 2 h after the application of EFs. Along with the increase in vimentin and the cadherin switch, these changes suggest that treatment with EFs causes the loss of epithelial characteristics and the acquisition of mesenchymal features. To further study specific expression changes associated with the EMT, we immunofluorescently stained EF-stimulated cells (6 h) for vimentin and E-cadherin expression, which are frequently used to monitor the progression of the EMT. Representative samples for each protein are shown in Fig. 1B. E-cadherin was positively expressed in almost all the cells but was suppressed after the EF treatment. Normal cells demonstrated either negative or mild staining for vimentin; meanwhile, EF-stimulated cells exhibited wide cytoplasmic staining for vimentin. These changes are in complete agreement with the corresponding Western blot results. Enhanced migratory capacity is a critical feature of the EMT that facilitates reepithelialization. Images recording cell trajectories showed that EF stimulation greatly increased their range of movement. The statistical analysis of the velocity confirmed the positive effect of the EF treatment on the EMT via increased motility (Fig. 1C). The effect of EFs on migratory capacity was further confirmed using scratch assays. As shown by the representatively results in Fig. 1D, cells stimulated by EFs migrated into nearly 67% of the original cell-free area while the area covered by normal cells was only 33% after 12 h. The statistical analysis of the monolayer migration assays demonstrated a positive correlation between the EF treatment and the enhanced migratory capacity, which was consistent with the single-cell motility results (P < 0.05). Taken together, these findings demonstrate that EFs equivalent in strength to physiological EFs found in vivo stimulate keratinocyte mobilization and gain of mesenchymal features. 3.2. EF-induced suppression of PTEN drives the EMT acquisition After verifying the occurrence of the EF-induced EMT, we speculated that there might be a signal relaying electrical cues into cellular responses. Notably, PTEN plays a critical role in these two processes, which prompted us to study the possibility that PTEN suppression is correlated with the EF-induced EMT. After demonstrating that no differences existed between normal cells and siNC-transfected cells (data not shown), we investigated
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whether EFs induce PTEN down regulation. As clearly presented in Fig. 2A, EFs suppressed the expression of PTEN in a timedependent manner. Then, the role of PTEN in the EF-induced EMT was evaluated by characterizing the effects of further downregulating PTEN expression using siPTEN and over-expressing PTEN expression using recombinant adenovirus vectors (AdPTEN). No changes in transcription factors or protein markers were observed in cells transfected with siNC and Vector. Meanwhile, cells treated with EFs exhibited EMT, with the loss-of-epithelial markers and gain-of-mesenchymal features. Further-decreased PTEN expression dramatically promoted the EF-induced EMT, as illustrated by the increased expression of Snail, Slug, vimentin and N-cadherin accompanied by the decreased E-cadherin expression (Fig. 2B). Similar to the Western blot results, the IF findings also showed that PTEN knockdown increased vimentin expression and decreased E-cadherin expression (Fig. 2C). As shown by the Western blot, PTEN overexpression inhibited EMT acquisition (Supplementary Fig. S1A). Additionally, IF results showed that PTEN overexpression under normal culture conditions induced higher E-cadherin expression (stronger relative fluorescence intensity, data not shown) and almost entirely abolished vimentin expression (lower relative fluorescence intensity, data not shown). PTEN overexpression in EFs-stimulated cells also increased Ecadherin expression and decreased vimentin expression, reflecting inhibition of EMT (Supplementary Fig. S1B). To determine whether PTEN is a mediator of EMT-associated migration, we measured the movement ranges and coverage areas in the cell motility and monolayer migration assays, respectively. As expected, the knockdown of PTEN further increased the movement range of cells compared with that of the siNCtransfected control cells (Fig. 2D). The siNC-transfected cells that had undergone the EMT after the EF pretreatment achieved 76% wound closure, and silencing PTEN expression resulted in nearly 95% coverage of the original wound (Fig. 2E). In addition, PTEN overexpression inhibited the single cell motility and monolayer migration of keratinocytes (Supplementary Fig. S1C and D). These results define PTEN as a regulator of the EMT in keratinocytes when exposed to EFs; how PTEN regulates the EMT requires further study. 3.3. mTORC1 is required for the EF-induced EMT and enhanced migratory capacity mTORC1 attracted our attention when studying how PTEN regulates the EMT since it is coupled with PTEN in underlying the cellular responses to EFs. After demonstrating that no differences existed between normal cells and cells treated with DMSO (data not shown), we first determined whether mTORC1 is involved in the EFs-induced responses and EMT. Phospho-p70S6 kinase (Thr389) and phospho-4E-BP1 (Thr70) serve as indicators of mTORC1 activity, which can be visualized using commercially available antibodies. In the time-course experiments, the levels of p70S6K and 4E-BP1 phosphorylation were dramatically increased with prolonged stimulation times. Total p70S6K and 4E-BP1 levels were not affected by the EF treatment (Fig. 3A). Rapamycin is a classical inhibitor of mTORC1 and was used to decipher the role mTORC1 plays in regulating the EMT. Cells incubated with Rapamycin demonstrated over a 90% reduction in phospho-p70S6K and phospho-4E-BP1 levels. Since the targeted inhibition of mTORC1 was confirmed, we explored whether its inhibition is sufficient to attenuate the EF-induced EMT by examining the expression levels of the aforementioned markers. As shown in Fig. 3B, the EF-induced induction of Snail, Slug, vimentin, and N-cadherin expression and decrease in E-cadherin expression were attenuated in keratinocytes with inhibited mTORC1. IF confirmed the increased levels of E-cadherin (stronger
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Fig. 1. EFs induce the EMT with enhanced motility and migration. (A) Western blot results of cells stimulated by EFs and probed for Snail, Slug, vimentin, E-cadherin, Ncadherin, b-actin (loading control). (B) IF staining for E-cadherin (green), vimentin (red) and the nuclear compartment (blue) in control and EF-stimulated cells. Scale bar = 25 mm. (C) The movement trajectories of cells in the control and EF-stimulated groups were recorded by a Zeiss imaging system. The velocity of each group is shown as the mean SD. *P < 0.05 versus the control group. (D) Scratch assays were used to confirm the migration-promoting effect of EFs in keratinocytes. The wound closure (%) was calculated by the reduction of the wound area over time and is shown as the mean SD. *P < 0.05 versus the control group. Scalebar = 200 mm.
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Fig. 2. EF-induced suppression of PTEN contributes to both the EMT and migratory capacity. (A) Immunoblot analysis for PTEN expression in control and EF-stimulated cells. (B) Immunoblot for the expression of PTEN and EMT markers in siRNA-transfected cells with or without EF treatment. (C) IF staining in control (transfected with siNC), siPTEN, 6-h EF-treated (transfected with siNC or siPTEN) cells. Scale bar = 25 mm. (D) The movement trajectories of control (transfected with siNC), siPTEN, 6-h EF-treated (transfected with siNC or siPTEN) cells. *P < 0.05 versus the control group. #P < 0.05 versus the 6-h group. (E) Monolayer migration in control (transfected with siNC), siPTEN, 6-h EFtreated (transfected with siNC or siPTEN) cells. *P < 0.05 versus the control group. #P < 0.05 versus the 6-h group. Scalebar = 200 mm.
relative fluorescence intensity, data not shown) and the decreased levels (nearly abolished) of vimentin (lower relative fluorescence intensity, data not shown) in mTORC1 inhibited groups (Rapamycin, 6h + Rapamycin) compared with the N group and 6-h group, respectively (Fig. 3 C). The migratory capacity of the cells above was assessed using a Zeiss imaging system and scratch assays. mTORC1 inhibition
significantly decreased the movement range of cells and velocity of the keratinocytes (Fig. 3D). Consistent with these results, mTORC1 inhibition significantly decreased the migration of cells exposed to EFs, as demonstrated using a wound-healing assay (Fig. 3E). Collectively, these data show that mTORC1 is both activated in EFtreated keratinocytes and required for the EMT, an important step in the initiation of reepithelialization during wound healing.
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Fig. 3. mTORC1 is required for EF-induced EMT and enhanced migratory capacity. (A) Western blot detecting the activity of mTORC1 in EF-treated cells through the expression levels of phosphor-p70S6K (Thr389), p70S6K, phosphor-4E-BP1 (Thr70), 4E-BP1, and b-actin (loading control). (B) Western blot demonstrating changes in mTORC1 activity and EMT markers levels in control and EF-stimulated cells treated with DMSO or Rapamycin. (C) IF staining for E-cadherin (green), vimentin (red) and the nuclear compartment (blue) in control cells (treated with DMSO or Rapamycin) and 6-h EF-stimulated cells treated with DMSO or Rapamycin. Scale bar = 25 mm. (D) The movement trajectories of control cells (treated with DMSO or Rapamycin) and 6-h EF-stimulated cells treated with DMSO or Rapamycin. *P < 0.05 versus the control group. #P < 0.05
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3.4. mTORC1 is involved in the occurrence of the EMT driven by PTEN suppression Our results show that PTEN suppression and mTORC1 activation are both required for gaining mesenchymal features and enhanced migratory capacity, which normally accompany the EMT. Considering the reports in the literatures demonstrating that PTEN can suppress mTORC1, we reasoned that during the EMT process, there might be a causal relationship between PTEN and mTORC1. To test this possibility, we first showed that no differences existed among normal cells and cells treated with DMSO, siNC or both (data not shown); then, we detected the activity of mTORC1 in cells after PTEN-knockdown and found that the levels of p70S6K and 4E-BP1 phosphorylation were further increased compared with those of cells exposed to EFs, verifying that PTEN can regulate mTORC1 activity. Meanwhile, the expression level of PTEN was not affected by mTORC1 inhibition. Consistently, the changes of the aforementioned EMT markers demonstrated that PTEN knockdown promoted the occurrence of the EMT in EF-stimulated cells. This PTEN suppression-related EMT was attenuated in cells with inhibited mTORC1, as shown in Fig. 4A, suggesting that mTORC1 is necessary for the occurrence of the EMT induced by PTEN suppression (Fig. 4A). Coincidentally, similar results were found by IF. EFstimulated cells showed downregulated E-cadherin compared to normal cells (data not shown), and siPTEN further reduced the expression of E-cadherin, which could be reversed by Rapamycin treatment. Similar results were also found via vimentin staining (Fig. 4B). We utilized a Zeiss imaging system and scratch assays to determine whether mTORC1 is essential for the enhanced migratory capacity of PTEN-knockdown cells. Cells treated with EFs had larger movement ranges than normal cells (data not shown), which was consistent with the results described above. PTEN knockdown further enlarged the movement range of cells treated with EFs. With the inhibition of both PTEN and mTORC1, the trajectories of cells were substantially decreased compared with the cells that were only targeted for PTEN knockdown (Fig. 4C). Cells that were stimulated by EFs and underwent the EMT achieved 70% wound closure. Meanwhile, the targeted knockdown of only PTEN resulted in nearly 95% coverage of the original cellfree area, while the additional inhibition of mTORC1 reversed the PTEN downregulation-induced migration, leading to only 40% wound coverage (Fig. 4D). Collectively, our results acquired by integrating multiple experimental approaches demonstrate that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation. 4. Discussion Upon injury, skin disruption instantaneously generates EFs due to the collapse of TEPs. Accumulating evidence indicates that EFs are vital and significant occurrences to which keratinocytes are subjected. To repair skin wounds effectively, keratinocytes from the wound margin undergo the EMT, which allows them to lose their epithelial characteristics and acquire mesenchymal features and migratory properties. However, related studies that have not considered these EFs may ultimately be unable to reflect the complexity of the EMT of keratinocytes as they respond to changing environments[24]. In this study, we determined the role of EFs in regulating the EMT in keratinocytes for the first time. First, we demonstrated that EFs induce the EMT along with enhanced migratory abilities, which normally accompany the EMT (Fig. 1).
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Second, we found that the EF-induced inhibition of PTEN and activation of mTORC1 are both greatly involved in the EMT (Figs. 2 and 3, Supplementary Fig. S1). These findings identify PTEN and mTORC1 as functional molecules in the EF-induced EMT and important regulators of EMT processes. Third, we showed that the inhibition of mTORC1 reversed the PTEN suppression-induced EMT and migratory capacity concomitant with attenuated EMT hallmarks (Fig. 4), suggesting that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation (Fig. 5). The EMT is a key reversible step that facilitates reepithelialization as well as tumor metastasis and invasion. Due to the clinical significance of this process, the induction of the EMT is an appealing therapeutic approach that could meaningfully expedite wound repair. Our findings implicate EFs as key regulators of the EMT in keratinocytes, which is achieved by PTEN modulation and mTORC1 activation. This conclusion is grounded on the observations that applied EFs induced a range of biochemical changes (e.g., increased expression of Snail, Slug, vimentin, N-cadherin, and decreased E-cadherin expression) and functional chang es (e.g., enhanced migratory capacity) that are characteristic of the EMT [12]. This new discovery further highlights the importance of EFs in wound healing, as the EMT enables keratinocytes to transform from sedentary cells into motile cells. In this context, we suggest a novel role for EFs in the support of wound repair by inducing the EMT. In normal skin, keratinocytes go through a process of terminal differentiation and provide a barrier that protects the underlying tissue from pathogenic microorganisms invasion and mechanical, chemical, and physical damage. The barrier function is enhanced by cell–substrate and cell–cell interactions, such as tight junctions and adherens junctions [25–28]. Upon wounding, the cells from the wound margin proceed through the EMT and transform from proliferative, sedentary cells into migratory cells; this process is essential for successfully achieving reepithelialization. During the EMT, Snail (also known as Snail1) and Slug (also known as Snail2), as transcription factors, repress the expression of cell junction proteins [29–31]. The downregulation of E-cadherin is balanced by the increased level of N-cadherin. Through this cadherin switch, the transitioning cells acquire N-cadherin interactions, which are weaker than E-cadherin interactions and facilitate cell motility and migration. Additionally, the induced expression of vimentin is closely related with the other EMT hallmarks and regulates both the membrane-associated protein activity and organelles trafficking. These changes are widely accepted biomarkers of the EMT. In our study, the fact that EFs were found to induce the EMT by studying the aforementioned markers raises a legitimate question: how are electrical cues relayed into cellular responses causing the EMT? PI3K and PTEN are key molecules in sensing electrical cues. EFs activate PI3K signaling in keratinocytes rapidly and specifically [9]. PTEN is a negative regulator of PI3K and has been shown to be downregulated in several cell types [22]. Therefore, its deletion enhances PI3K activation and cellular reactivity to EFs [1]. Thus, PI3K and PTEN represent molecules and genes underlying cellular responses to EFs. The mTOR signaling pathway is an important component of PI3K signaling, and it integrates multiple types of information, such as the levels of energy, growth factors and hypoxia, to facilitate the migration and invasion of cancer cells. While intensive studies have focused on the role of mTORC1 in regulating the EMT, growth, survival, and motility of cancer cells under normal conditions, its role in EF-treated keratinocytes and the related cellular responses has not been extensively studied. We found that the pharmacological inhibition of mTORC1 significantly
versus the 6-h group. (E) Monolayer migration in control cells (treated with DMSO or Rapamycin) and 6-h EF-stimulated cells treated with DMSO or Rapamycin. *P < 0.05 versus the control group. #P < 0.05 versus the 6-h group. Scalebar = 200 mm.
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Fig. 4. mTORC1 is involved in the acquisition of EMT driven by PTEN suppression. (A) Western blot results for cells treated with siNC, siPTEN, DMSO or Rapamycin detecting the expression levels of PTEN, phosphor-p70S6K (Thr389), p70S6 K, phosphor-4E-BP1 (Thr70), 4E-BP1 and EMT markers. (B) IF staining for vimentin, E-cadherin and the nuclear compartment in groups treated as described in (A). Scale bar = 25 mm. (C) The movement trajectories of groups treated as described in (A) were recorded and analyzed. *P < 0.05 versus the 6-h group. #P < 0.05 versus the siPTEN group. (D) The scratch assay results, represented as wound closure, of the group treated as described in (A) were recorded and analyzed and are shown as the mean SD. *P < 0.05 versus the 6-h group. #P < 0.05 versus the siPTEN group. Scalebar = 200 mm.
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Fig. 5. Schematic illustrating that the EF-induced suppression of PTEN drives the EMT via mTORC1 activation during wound healing.
reversed EF-induced and PTEN-knockdown-related EMT, which was accompanied by attenuated migratory ability. Our results are consistent with those of previous studies demonstrating that the inhibition of mTORC1 in various cancer types potently inhibited the EMT and motility induced by TGFb, IGF-1 and EGF [12,32–34], suggesting that multiple signals ultimately converge upon mTORC1. As a negative regulator, PTEN deletion or knockdown has also been demonstrated to activate mTORC1 and facilitate improved motility and EMT acquisition. These discoveries highlight the fact that these functions of PTEN and its interaction partners are not cell-type specific and occur in various types of circumstances and cells. However, our study is just a preliminary exploration of the potential signal network underlying EFs and the EMT, and these findings have raised many intriguing questions. How is PTEN downregulated by EFs? Do any other molecules sense and relay electrical cues into cellular responses causing the EMT? How does mTORC1 link the expression of transcription factors and changes in typical protein markers? Is there crosstalk among other important components of the PI3K pathway, such as mTORC2? Future studies may further elucidate this powerful component of wound healing and regeneration. Endogenous EFs are an intrinsic property of wounds. Clinically, the manipulation of EFs is a new and appealing avenue for improving and accelerating wound healing. Alternatively, pharmacologically enhancing the naturally occurring EFs could serve as a potent and promising approach toward developing wound-healing therapies. For instance, ascorbic acid and AgNO3 can activate Na+ channels and increase the TEPs and EFs [35–38]. Such drugs that converge on one common denominator, i.e., altered EFs, appear to be good candidates for accelerating wound healing. In conclusion, we have demonstrated for the first time that EFinduced PTEN repression drives the EMT via mTORC1 activation. These results provide a novel perspective of the significance of EFs in wound healing, which could lead to new wound management strategies. Author contributions T.Y., Y.H., X.J. developed the initial concept. Y.H. and X.J. designed the experiments, T.Y., X.G., W.C., D.T., J.Z., X.Z., Q.Z. and J.J. performed the research and analyzed the data. T.Y., D.Z., Y.H., D.T.
wrote the manuscript; Y.H. supervised the study. All authors discussed the results and commented on the manuscript. Conflict of interests The authors have no conflicts of interest to declare. Acknowledgements This work was supported by State Key Development Program for Basic Research of China (grant number 2012CB518101) and Research fund of the State Key laboratory of Trauma, Burns and Combined injury (grant numbers SKLZZ201203; SKLZZ2012(III)01). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2016.11.007. References [1] M. Zhao, B. Song, J. Pu, T. Wada, B. Reid, G. Tai, et al., Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN, Nature 442 (2006) 457–460. [2] M. Zhao, Electrical fields in wound healing–an overriding signal that directs cell migration, Semin. Cell Dev. Biol. 20 (2009) 674–682. [3] X. Guo, X. Jiang, X. Ren, H. Sun, D.Q. Zhang, et al., The galvanotactic migration of keratinocytes is enhanced by hypoxic preconditioning, Sci. Rep. 5 (2015) 10289. [4] M. Chiang, K.R. Robinson, J.W. Vanable Jr., Electrical fields in the vicinity of epithelial wounds in the isolated bovine eye, Exp. Eye Res. 54 (1992) 999– 1003. [5] A.T. Barker, L.F. Jaffe, J.W. Vanable Jr., The glabrous epidermis of cavies contains a powerful battery, Am. J. Physiol. 242 (1982) R358–366. [6] M. Zhao, J. Penninger, R.R. Isseroff, Electrical activation of wound-healing pathways, Adv. Skin Wound Care 1 (2010) 567–573. [7] C. Yan, W.A. Grimm, W.L. Garner, L. Qin, T. Travis, N. Tan, et al., Epithelial to mesenchymal transition in human skin wound healing is induced by tumor necrosis factor-alpha through bone morphogenic protein-2, Am. J. Pathol. 176 (2010) 2247–2258. [8] M. Nakamura, Y. Tokura, Epithelial-mesenchymal transition in the skin, J. Dermatol. Sci. 61 (2011) 7–13. [9] P. Martin, Wound healing–aiming for perfect skin regeneration, Science 276 (1997) 75–81. [10] M.C. Heng, Wound healing in adult skin: aiming for perfect regeneration, Int. J. Dermatol. 50 (2011) 1058–1066.
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Please cite this article in press as: T. Yan, et al., Electric field-induced suppression of PTEN drives epithelial-to-mesenchymal transition via mTORC1 activation, J Dermatol Sci (2016), http://dx.doi.org/10.1016/j.jdermsci.2016.11.007