Polarization of ADAM17‐driven EGFR signalling in electric field‐guided collective migration of epidermal sheets

Abstract Endogenous electric field is considered to play an important role in promoting collective migration of epidermis to the wound centre. However, most studies are focused on the effect of bioelectric field on the movement and migration of single epithelial cell; the molecular mechanisms about collective migration of epidermal monolayers remain unclear. Here, we found that EFs dramatically promoted the collective migration of HaCaT cells towards the anode, activated the sheddase activity of ADAM17 and increased the phosphorylation level of EGFR. Moreover, EGFR phosphorylation and HB‐EGF shedding level were significantly decreased by the ADAM17 inhibitor TAPI‐2 or siADAM17 under EFs, which subsequently attenuated the directed migration of HaCaT sheets. Notably, the inhibition of EF‐regulated collective migration by siADAM17 was rescued by addition of recombinant HB‐EGF. Furthermore, we observed that F‐actin was dynamically polarized along the leading edge of the migrated sheets under EFs and that this polarization was regulated by ADAM17/HB‐EGF/EGFR signalling. In conclusion, our study indicated that ADAM17 contributed to the collective directional movement of the epidermal monolayer by driving HB‐EGF release and activating EGFR under EFs, and this pathway also mediated the polarization of F‐actin in migrating sheets, which is essential in directional migration.


Abstract
Endogenous electric field is considered to play an important role in promoting collective migration of epidermis to the wound centre. However, most studies are focused on the effect of bioelectric field on the movement and migration of single epithelial cell; the molecular mechanisms about collective migration of epidermal monolayers remain unclear. Here, we found that EFs dramatically promoted the collective migration of HaCaT cells towards the anode, activated the sheddase activity of ADAM17 and increased the phosphorylation level of EGFR. Moreover, EGFR phosphorylation and HB-EGF shedding level were significantly decreased by the ADAM17 inhibitor TAPI-2 or siADAM17 under EFs, which subsequently attenuated the directed migration of HaCaT sheets. Notably, the inhibition of EF-regulated collective migration by siADAM17 was rescued by addition of recombinant HB-EGF. Furthermore, we observed that F-actin was dynamically polarized along the leading edge of the migrated sheets under EFs and that this polarization was regulated by ADAM17/HB-EGF/EGFR signalling. In conclusion, our study indicated that ADAM17 contributed to the collective directional movement of the epidermal monolayer by driving HB-EGF release and activating EGFR under EFs, and this pathway also mediated the polarization of F-actin in migrating sheets, which is essential in directional migration.

K E Y W O R D S
ADAM17, collective directional migration, EGFR, electric fields, F-actin, HB-EGF, wound healing centre to become more negative than the surrounding area, and the electric signals can override other cues to promote electrotactic responses, a phenomenon called cell electrotaxis/galvanotaxis. 3 Numerous in vitro experiments confirmed the EF-induced directional migration in many cell types such as corneal epithelial cells, endothelial cells, keratinocytes, and fibroblasts. A number of researchers investigated the biomolecular intracellular signalling pathways to reveal how the cells sense and control the polarity in response to the directional electric cue at a single-cell level.
The intracellular 'compass model' suggests a competition between the PI3K-dependent pathway at the front and the myosin-dependent pathway at the rear of the cell that determines the direction of single-cell migration by the active formation of lamellipodia in directional response to the applied EF. 4 EF was shown to induce a polarized activation of several other signalling pathways such as phosphatase and tensin homologue (PTEN), epidermal growth factor (EGF) receptors, mitogen-activated protein kinase (MAPK), extracellular-signal-regulated kinase (ERK) and RhoA. 5 The limitation of current knowledge is that the studies on the electrotactic response dealt with the cells that are in isolation.
However, cellular motility in wound conditions concerns with the collective migration and it is unclear how an electric field will affect collective migration. Severe previous studies have demonstrated polarization of cell surface EGFR that coincides with the direction of electrotactic cell migration. 6 In addition, EGFR has been implicated in regulating galvanotaxis in several cell populations, including fibroblasts, keratinocytes and other epithelial cells. 7 In human keratinocytes, it has been reported that EGFR polarization occurred as early as 5 minutes after exposure to electric fields; inhibition of EGFR abolished not only the EGFR redistribution, but also the electrotactic migration. 8 EF induced EGFR phosphorylation in a ligand-independent manner, and subsequent inhibition of EGFR activity restrained galvanotaxis. Although EGFR is functional in collective migration, and more importantly, how it is activated by EFs has not been identified yet.
Recently, ADAM17 has been proved as the principal activators of the EGFR activation during wound repair. ADAM17 was identified as a major sheddase responsible for the ectodomain shedding of the multiple membrane-bound EGFR ligand precursor, including heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α) and amphiregulin (AREG) that activate epidermal growth factor receptor (EGFR), which plays a significant role in wound healing. 9 Thus, it is possible that EGF/EGFR pathway regulated by ADAM17 and thereby conducing directional migration of epithelial sheets.
The impact of ADAM17 on the directional collective migration of the epidermal monolayer by the application of physiological-level EFs has not been assessed. We assume that directional collective migration of epidermal sheets under EFs is associated with increased activity of ADAM17 and thereby activation of HB-EGF/EGFR signalling pathway. In the current study, we demonstrate that the HaCaT monolayer shows marked collective electrotactic migration and that its electrotactic response is regulated by ADAM17. This effect is dependent on activation of the HB-EGF/EGFR signalling pathway.
We also show that EF-inducing F-actin polarization is mediated by the ADAM17/HB-EGF/EGFR signalling pathway, is consistent with directional migration of epidermal monolayer.

| Cell culture
The HaCaT cells were obtained from the Cell Bank of the Chinese Academy of Sciences in Beijing, China. Cells were cultured in RPMI 1640 medium supplemented with 100 μg/ml streptomycin, 100 U/ ml penicillin and 10% foetal bovine serum (FBS). The HaCaT cells were incubated at 37°C in 95% humidity and 5% CO 2 .

| EF stimulating and time-lapse image recording
To observe HaCaT cells migration, the previously developed experimental electrotaxis chamber was utilized, as described. 10,11 To stimulate cells, EFs were fabricated through two silver electrodes immersed in Steinberg's solution-filled reservoirs, which were connected to the culture medium by two agar bridges (2% agar in Steinberg's solution). Before EF stimulation, a CO2-independent culture medium was transferred to the prepared chamber. Cell monolayer was exposed to EFs with electrical strengths ranging from 0 to 200 mV/mm for 6 h. Time-lapse imaging was performed with a Zeiss imaging system (Carl Zeiss Meditec, Jena, Germany) to monitor migration of the cell monolayer, and images were recorded every 5 min for 6 h. Images analysis were implemented by using NIH ImageJ software.

| Quantitative analysis of cell migration velocity and directedness
Directional cell migration was quantified using methods reported previously. 10,12 The directedness of cells was quantified by cos θ, which represents the angle between the field vector and the cell migration direction. Trajectory speed (Tt/t) was used to quantify the x-axis was calculated as the cell's displacement distance along the EF vector divided by the period of time. The relative fluorescence of internally cleaved products was measured at an excitation wavelength of ~324 nm and emission wavelength of ~410 nm. All data were normalized with respect to data from the control group.

| Western blot analysis
The cell monolayer was washed with ice-cold phosphate-buffered sa- membranes. The membranes were blocked in 5% bovine serum albumin (BSA) (Sigma-Aldrich) in TBST for 3 hours at room temperature.
The blots were probed with primary antibodies at 4°C overnight. The above primary antibodies were used at a 1:1000 dilution, the loading control anti-β-actin at a 1:4000 dilution and the secondary antibody at a 1:4000 dilution. The membranes were probed using the following primary antibodies: anti-β-actin (Proteintech, USA), anti-ADAM17 (Abcam ab39162, UK), anti-EGFR (Abcam ab52894, UK) and anti-p-EGFR (Abcam ab40815, UK). The images were quantified with the Quantity One 4.1 software (Bio-Rad, USA).

| ELISA
To evaluate the release of ADAM17 substrates, the cell monolayer was stimulated by an EF for 6 h, and then, the supernatant was collected. Relevant DuoSet ELISA kits (Cloud-clone Corp, HB-EGF SEB479Hu, AREG SEA006Hu, TGF-α SEA123Hu, China) were adopted to analyse the supernatant.

| Immunofluorescence
Following EF-directed HaCaT cells migration, the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature.
After washing with PBS three times, the cell monolayer was blocked with 5% BSA (Sigma-Aldrich). Staining F-actin with rhodamine phalloidin (Proteintech, USA) at a 1:50 dilution for 2 hours. After washing with PBS, the cell monolayer was mounted with DAPI (Sigma F6057, USA). Images were obtained using a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).

| Statistical analysis
The values are expressed as the mean ± SEM. The results were compared by analysis of variance for repeated measurements, followed by Student-Newman-Keuls t test. Comparisons in different groups were performed by using Tukey's honestly significant difference (HSD) test or Dunnett's test. P < .05 was considered statistically significant.

| Collective electrotaxis of epithelial monolayers under EF
To evaluate the electrotactic behaviour of the epidermal monolayer, we first observed EF-stimulated migration of the HaCaT cells by time-lapse microscopy in the absence or presence of an EF. In the absence of an EF, no obvious directional migration was observed.
The cells in a confluent monolayer migrated very little and randomly.
In an EF of 100 mV/mm, the cells migrated towards the anode collectively, at an increased speed ( Figure 1A, Movie S1). Although the cell monolayer migrated collectively and directionally, the single cell maintained their relative position in a monolayer, and the leading cells were seen to extend lamellipodia and filopodia towards the anode (Movie S1). This directional response, as indicated by the value of directedness, could be detected at 30 minutes, peaking at ( Figure 1C-D) (P < .01, when compared with No EF control). The threshold voltage required to induce electrotaxis of cell monolayers was therefore largely lower than 50 mV/mm. Applied EFs also significantly increased the trajectory speed. The increase in speed was also dependent on field strength, with an EF of 200 mV/mm increasing the speed by 231% ( Figure 1E). Consistent with the directional migration, displacement along the χ-axis (the field line towards the anode) in cells also significantly increased following EFs stimulation ( Figure 1F-G). These results suggest a robust electrotactic response in epithelial monolayer in a time-and voltage-dependent manner.

| ADAM17 is required for EF-induced collective directional migration of the epidermal monolayer
To determine whether ADAM17 contributes to the collective directional migration of epidermal monolayers under EFs, we first examined the enzymatic activity and distribution of ADAM17 in a HaCaT monolayer after EFs application. As shown in Figure  Compared with No EF group, the ADAM17 polarization towards the anode increased significantly from 9% to 80% under EFs at the leading edge ( Figure 2B,C). These results suggest that EFs have significant effects on ADAM17.
Next, we examined a possible role of ADAM 17 in EF-induced collective directional migration of HaCaT cell monolayer by pretreatment of TAPI-2, a specific ADAM17 inhibitor or siADAM17, a specific ADAM17-small interference RNA. Immunofluorescence staining and immunoblot analysis all confirmed a complete depletion of ADAM17 by siADAM17 transfection ( Figure S2). Strikingly, we found that both TAPI-2 and si-ADAM17 caused profound reductions in EF-induced directional collective migration of cells ( Figure 2D,E; Movie S3-S4). The migration directedness in TAPI-2 and si-ADAM17 pretreated monolayers declined by 74% and 80%, respectively, when compared to controls ( Figure 2F). Trajectory

| ADAM17 contributes to EF-directed collective migration through EGFR signalling
As ADAM17 has been proven as a major sheddase responsible for the ectodomain shedding of the multiple membrane-bound EGFR ligand precursor, and EGFR has been identified to control the electrotactic response of epithelial cells, we then hypothesized that ADAM17 may contribute to the EF-induced collective directional migration of the cells via EGFR. To test our hypothesis, we firstly examined the status of EGFR activation (tyrosine-phosphorylated EGFR) during ADAM17-regulated electrotactic responses of the epidermal monolayer. As shown in Figure 3A, EGFR phosphorylation was strongly increased in EF-treated monolayer as compared to No EF control. The EF-induced EGFR phosphorylation could be totally abolished by TAPI-2 or siADAM17 pretreatment, indicating a crucial role for ADAM17 in EF-induced EGFR activation ( Figure 3B,C).

F I G U R E 3 ADAM17-induced EGFR activation promotes the collective directional migration of the epidermal monolayer under EFs. A,
Representative immunoblotting and quantification of p-EGFR in HaCaT monolayer exposed to EFs. B, Representative immunoblotting and quantification of p-EGFR in HaCaT monolayer exposed to TAPI-2 under EFs. C, Representative immunoblotting and quantification of p-EGFR in HaCaT monolayer exposed to si-ADAM17 under EFs. D, Fluorescence confocal images showing the staining and distribution of EGFR after EF exposure without drugs or with siRNA-ADAM17 treatment. E, Statistical analysis indicating the EGFR distribution after EF exposure without drugs or with siRNA-ADAM17 treatment. F, Representative images of HaCaT monolayer at 6 h after EF stimulation in various groups. G, Migration trajectories of HaCaT monolayer at 6 h after EF stimulation in various groups. H-I, Effect of the EGFR inhibitor AG1478 on migration directedness and trajectory speed. Arrow indicates the direction of the electric field. Composite images (D) are merged images consisting of 2 channels: EGFR (red) and DAPI (blue). Data were obtained from at least three independent experiments and are shown as the mean ± SEM. #P < .05 vs. the EF group. Scale bars (D) 10 µm, (F) 50 μm Morphologically, we also found that EGFR showed a strong staining in the extended lamellipodia and filopodia at the anodal side of the leading cells after exposure to the electric field ( Figure 3D), similar to the ADAM 17 staining under EFs ( Figure 2B). This polarization staining of EGFR could be totally disrupted by siADAM17 pretreated, indicating that not only the activation, but also the asymmetrical redistribution of EGFR under EFs was controlled by ADAM17 ( Figure 3D,E). Accordingly, we observed an obvious reduction in the collective electrotactic response in HaCaT monolayer exposed to AG1478, an EGFR tyrosine kinase inhibitor ( Figure 3F,G; Movie S5). With exposure to EGFR inhibitor, the mean directedness of cells decreased from −0.88 to −0.08 ( Figure 3H), and the mean trajectory speed decreased from 0.30 μm/min to 0.18 μm/min ( Figure 3I).
Inhibition of EGFR virtually abolished the electrotactic response of epithelial sheets. These data suggest that ADAM17 facilitates the EF-induced collective directional migration of cells through EGFR signalling.

| HB-EGF plays a key role in ADAM17/EGFR axis-mediated collective directional migration under EFs
In ADAM17 active epidermis, EGF family molecules such as HB-EGF, TGF-α and amphiregulin (AREG) are released, which contribute to the re-epidermalization and accelerates wound healing.
To identify which EGF molecules play a key role in the ADAM17/ EGFR axis-mediated collective directional migration under EFs, we examined the release of EGF molecules in HaCaT cell monolayer  Figure 4H). These results, taken together, suggest that HB-EGF plays a pivotal role in ADAM17/EGFR axis-mediated collective electrotactic migration in epidermal monolayer.

| F-actin polarization is controlled by the ADAM17/EGFR axis in epidermal monolayer exposed to EFs
Cell migration is mediated by the protrusion of cytoplasm termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signalling complexes. The importance of F-actin polarization at the leading edge of migrating sheets in the initiation and maintenance of the EF-directed collective migration has been confirmed in epidermal cells. 13 Here, we investigated whether the EF-directed F-actin polarization is regulated by the ADAM17/EGFR axis. As compared to No EF control,

| D ISCUSS I ON
In this study, we demonstrated that EF promotes the directional migration of epidermal sheets through the activation and polarization of ADAM17/EGFR axis. In this axis, HB-EGF, controlled by ADAM17 shedding, leads to EGFR activation and the subsequent F-actin polarization, gearing up a directional collective migration of epidermal monolayer ( Figure 6). EFs. 17,18 In our study, we did find that EF at strength as low as   ing, and trigger ADAM17-mediated shedding of its substrates from the cell surface. 28 The above researches provide a new enlightenment for us to further explore the mechanism of ADAM17 activation by electric field.
Effective directionality requires cooperation of numerous cellular movements such as the formation of lamellipodia and filopodia at the leading edge. 29 The formation of lamellipodia and filopodia is driven by actin polymerization, a process regulated by signalling complexes. Previous work has shown that ADAM17 up-regulation enhances actin cytoskeletal remodelling at the tip of the lamellipodium in HCC cells. 30 Studies from Zhao and Fang et al have indicated a crucial role for HB-EGF/EGFR signalling in F-actin colocalization and polarization in a physiological EF. 8,13 In our study, we observed the polarization of ADAM17, EGFR and F-actin in the electrotactic collective migration of cell sheets ( Figures 2B, 3D and 5B). EGFR inhibition or ADAM17 silencing prohibited cell protrusions and F-actin redistribution, along with the reduction of the electrotactic collective migration ( Figure 5). These data provided F I G U R E 6 Schematic model depicting the role of ADAM17/HB-EGF/EGFR signalling pathway in directional collective migration of epidermal cells cytoskeletal evidence further confirming the involvement of ADAM17/EGFR signalling axis in EF-guided collective migration.
In conclusion, our findings indicate that EFs guide a robust directional collective migration in epidermal sheets. Moreover, ADAM17 is responsible for this obvious EF-induced collective directional migration. Importantly, the above electrotactic migration mechanism is regulated by the HB-EGF/EGFR signalling pathway. F-actin is significantly redistributed towards the leading edge of migrating sheets, and F-actin polarization is linked to the ADAM17/HB-EGF/ EGFR signalling pathway. Our study reveals novel findings related to the signalling mechanism of EF-guided collective migration of epithelial cells, which could lead to new wound management strategies.

ACK N OWLED G EM ENTS
This work was supported by the National Nature Science Foundation of China (NSFC No. 81873936).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial or non-financial interests.