The Mechanism Of ADAM17 Regulates Electric Fields-mediated Collective Directional Migration Of Epidermal Monolayer

BackgroundWound healing involves several epithelial cells functions,such as proliferation,migration,and differentiation,and the migration of epithelial cells towards the wound centre is a pivotal step.It has been shown that epithelial cells migrate as a coherent monolayer to accelerate wound healing.Impaired ability of the epithelial sheets to migrate across the damage site is characteristic of chronic non-healing wounds.In order to migrate effectively to repair the wounds,epithelial sheets must know not only when to direct,but also,very importantly,in which direction.Endogenous electric fields(EFs),an overriding other guidance cues,guided directional collective movement of epithelial cells during heling process.In wounds of epithelial tissue,endogenous EFs are naturally generated due to the collapse of transepidermal potentials(TEPs),causing the wounded centre to become more negative than the surrounding area,and the electric signals induced electrotactic responses of cells,a phenomenon called electrotaxis/galvanotaxis.In vitro,our previously demonstrated that keratinocytes exhibit electrotactic response by applied EFs at a strength equivalent to those measured in vivo(40–200 m V/mm).Effective electrotaxis is always accompanied by the generation of cell polarization,and the membrane protrusion formed by actin on the leading edge of the migrating cell sheet is the main driving force for directional migration.Therefore,this electric signal serves as an essential cue in polarization and directional migration of epithelial monolayer.However,virtually nothing is known about the molecular mechanisms that mediate EF-induced polarization and directionality of epidermal monolayer.Importantly,our previous research shows a key regulator of A Disintegrin And Methalloprotease 17(ADAM17)in wound healing: it set up the initial origin for the cell migration of epithelial cells,and consequently activates HB-EGF/EGFR signalling,which is necessary to accelerate wound re-epithelialization.ADAM17 was identified as a major sheddase responsible for the ectodomain shedding of HB-EGF,which plays a significant role in the resolution of epithelial inflammation and tissue damage.Recently,many studies have reported that EGFR ligand,especially HB-EGF,greatly promote epithelial cell migration and significantly accelerate early wound healing reactions.Most importantly,EGFR is considered to be an important molecule involved in the electrotactic migration of cells.EGFR isinvolved in the electrotactic migration of a variety of cells,including fibroblasts,keratinocytes,and corneal epithelial cells.Previous studies showed that EF induced EGFR phosphorylation in a ligand-independent manner,and subsequent inhibition of EGFR activity restrained galvanotaxis.EGFR and F-actin colocalization and polarization toward the leading edge in a physiological EF.Specificlly,HB-EGF/EGFR pathway is required for cytoskeletal rearrangement,EGFR and F-actin colocalization and polarization in a physiological EF.Most importantly,However,whether ADAM17 plays a role in the electric field-induced polarization and collective directed migration of epidermal cells,and whether this regulation is regulated by the HB-EGF/EGFR signaling pathway is still unclear.Therefore,this study will explore the role and mechanism of ADAM17 in the electric fields to regulate the collective directed migration of epidermal cells.This study proposes that "the electric field accelerates the release of HB-EGF molecules to promote EGFR phosphorylation by activating ADAM17,thereby regulating the collective directed migration of epidermal cells.And this signal pathway also plays an important role in the polarity distribution of the cytoskeleton induced by electric fields.The scientific hypothesis has been proved through a series of experiments.The research is of great significance to deeply reveal the mechanism of ADAM17 involvement in wound epidermal migration and deepen the exploration of clinical treatment of wound healing.Contents and methods:1.Influence of EFs on collective directed migration of epidermal cellsAn experimental model for electrotaxis of epidermal cell sheets under external EF.Observe the characteristics of the electrotactic migration of epidermal monolayer and isolated cells in different field strengths.2.The role of ADAM17 on electric field-induced collective directional migration of epidermal cell monolayerThe ADAM17 activity test kit was used to test the changes of ADAM17 activity in epidermal cells in different electric field strengths.Immunofluorescence was used to observe the effect of the electric field on the distribution of ADAM17 in the leading edge of epidermal monolayer;The pretreatment of ADAM17 specific inhibitor TAPI-2 or transfection of ADAM17-siRNA into epidermal cells)affects the epithelial migration of epidermal cell sheets,and the role of ADAM17 in the epithelial migration of epidermal cells is clearly defined.3.The signaling mechanism of ADAM17 regulated-electrotactic response of epidermal cellmonolayerObserve the changes of HB-EGF release,EGFR phosphorylation and EGFR distribution in epidermal cell monolayer under the electric field;clearly inhibit ADAM17 activity or inhibit EGFR phosphorylation(EGFR-specific inhibitor AG1478)to release HB-EGF in epidermal cell sheet EGFR phosphorylation and distribution,as well as the effect of cell electrotactic migration;observe the effect of exogenous recombinant HB-EGF on ADAM17-siRNA pretreatment epidermal cell layer EGFR phosphorylation and electrotaxis.Reveal the pathway of HB-EGF/EGFR in ADAM17 regulated the electrotactic migration of epithelial monolayer.4.Effects of ADAM17/HB-EGF/EGFR signaling pathway on electric field-induced F-actin polarity distributionAfter applied the electric fields,immunofluorescence was used to observe the F-actin distribution in the leading edge of epidermal monolayer.On this basis,Observed the effect of inhibited ADAM17 or inhibited EGFR phosphorylation on the F-actin distribution in the leading edge of epidermal monolayer.Observe the exogenous recombinant HB-EGF on F-actin polarization in ADAM17-si RNA pretreatment epidermal sheets.Reveal the important signaling pathway of ADAM17/HB-EGF/EGFR in the electric field-induced actin cytoskeletion on polarization.Results1.In the presence of EF,HaCaT cell monolayer migrated towards the anode,the migration directedness and speed correlate strongly with the field strength.Epithelial monolayer exhibited more excellent electrotactic response than isolated cells,2.The electric field promotes the activation of ADAM17 with a voltage-dependent manner,and EF induced ADAM17 polarization towards the leading adge of epithelial sheets showing a polar distribution characteristic.Both TAPI-2 treatment and si-ADAM17 transfection triggered a profound reduction in the EF-induced directional collective migration.3.After applied the EFs,the release of HB-EGF is increased in epidermal monolayer,EGFR phosphorylation is enhanced,and EGFR polarization towards the leading edge of migrating sheets,showing showing a polarization characteristic of EGFR.Inhibition of ADAM17 activity or silencing of ADAM17 expression can significantly inhibit the electric field-induced HB-EGF release,EGFR phosphorylation and its polarization.Inhibition of EGFR significantly inhibited the electrotaxis migration of epidermal sheets.Exogenous recombinant HB-EGF can reverse the phosphorylation of EGFR caused by ADAM17-siRNA and the electrotactic response of epithelial sheet.4.The electric field induces F-actin toward the pseudopods of leading edge in epidermal monolayer,that is,the F-actin polarization occurs under the electric field.This polarization was abolished by ADAM17-siRNA and EGFR inhibitors.Exogenous recombination of HB-EGF can reverse the effect of ADAM17-siRNA on F-actin distribution,so that F-actin exhibits polarization characteristics.ConclusionIn conclusion,our findings indicate that the EFs guided more robust epidermal monolayer directional collective migration in wound healing process.Moreover,ADAM17 is responsible for this obvious EF-induced collective directional migration.Importantly,the above electrotactic migration is regulated by HB-EGF/EGFR signaling pathway.In addition,F-actin was re-distributed towards the leading edge of the migrated sheets,and this polymerization was linked to ADAM17/HB-EGF/EGFR signaling pathway,is consistent with directional migration of HaCaT sheets.Taken together,our studies provided novel evidence in signaling mechanism of EF-guided collective migration of epithelial cells,which could lead to new wound management strategies.