The Role Of CD9 In Electric Field-induced Collective Directional Migration Of Epidermal Cell Monolayers And The Signaling Mechanism

Background and purpose of the study:A fundamental feature of wound healing is the restoration of an intact epidermal barrier through wound epithelialization,also known as re-epithelialization.The collective directional migration of new epidermal cells from the trauma margin to the center of the wound is an important process of wound reepithelialization,and blockage and defects in this process will result in chronic refractory wound healing.Chemical and physical factors in the wound microenvironment play an important role in inducing the collective targeted migration of epidermal cells to the wound center;among them,wound endogenous electric fields(EFs)are considered to play an indispensable and important role.Due to the disruption of the transepithelial electrical potential(TEP)of the epidermis,wound endogenous electric fields are generated instantaneously after skin wound formation,resulting in a more negative electrical potential in the center of the wound than in the surrounding area,creating an electric field pointing from the periwound area to the center of the wound with a magnitude of approximately 42-200 mV/mm.Studies have shown that the electrical signal can direct the collective directional migration of epithelial cells,which is referred to as electrotaxis.In vitro studies have found that monolayers of cultured sheets of epidermal cells have a significantly better electrotaxis response than individually cultured epidermal cells.However,it remains largely unknown how electric fields direct the collective directional migration of cell monolayers and how this effect is more effective than that of isolated cells.Collective directional cell migration involves the organic coordination between two functionally distinct cell populations including leader cells(LC)and follower cells(FC).The leader cells sense migratory directional signals that lead to the formation of directional pseudopods with actin-based structures,which in turn play an important role in initiating and maintaining collective directional migration.CD9 is a member of a superfamily of four transmembrane proteins.It has been demonstrated that CD9 can co-localize with the cytoskeleton F-actin in filamentous pseudopods on the cell surface and has a role in regulating the actin cytoskeleton arrangement.However,in contrast to many other membrane surface proteins,CD9 does not function as a receptor or kinase.Studies have shown that CD9 is often required to bind or interact with membrane proteins such as signaling molecules receptors,a disintegrin and metalloprotease domains(ADAMs)to mediate a variety of physiological and cellular processes.Our preliminary study revealed that CD9 and a disintegrin and metalloprotease domain17(ADAM17)co-localize on the membrane surface of epidermal cells and confirmed the important role of CD9 in negatively regulating the sheddase activity of ADAM17,a known precursor sheddase for EGFR ligands such as HB-EGF,and was found to enhance actin cytoskeletal remodeling at the basal tip of the HCC cell plate.Our previous study also revealed that ADAM17 plays an important role in electric field-driven epidermal cell monolayer directed collective migration through activation of the HB-EGF/EGFR signaling axis.However,the mechanisms that induce and control leader cell polarization in electric field-driven epidermal monolayer collective migration remain to be elucidated.In addition,intercellular communication is considered to be crucial to improve the efficiency of collective migration,and paracrine secretion is a major form of intercellular communication through which cells respond to factors produced by nearby cells.Studies have shown that HB-EGF plays multiple functions in cell proliferation,migration and inflammatory responses through paracrine action.However,whether such paracrine mechanisms exist among leader cells to coordinate their polarization and migration behavior under electric fields,thus enhancing the electrotropism of collective cell migration,needs further investigation.In summary,we propose the scientific hypothesis that electric fields promote the activation of ADAM17/HB-EGF/EGFR signaling by down-regulating the expression of membrane molecule CD9,which induces the polar distribution of F-actin and pseudopod formation,thereby mediating the collective directional migration of epidermal monolayers,and test it using an experimental protocol.The study aims to reveal the molecular mechanism of collective directional migration of epidermal cells induced by electric field,which not only provides theoretical support for the clinical application of electric field technology,but also may provide potential new targets for trauma treatment.Research content and methods:1.Electric field induced collective directional migration of epidermal cell monolayers and its intrinsic relationship with F-actin polarization of leader cellsFor the first time,a cell electrotaxis model was used to observe the collective electrotactic response of epidermal cell(HaCaT)monolayers;secondly,the F-actin polarization and pseudopod formation of leader cells in epidermal monolayers in collective directional migration under electric field conditions were observed;Finally,the effect of inhibition of F-actin polymerization by cytochalasin B on F-actin polarization,pseudopodia formation of leader cells and collective directed migration induced by electric field was observed,and the internal relationship between F-actin polarization of leader cells and collective directed migration was clarified.2.Role of CD9 in electric field-induced F-actin polarization of leader cells and collective directional migration of epidermal cell monolayersFirstly,the effects of electric field on CD9 expression and distribution in epidermal cell monolayers were observed by immunofluorescence and Western Blot experiments;secondly,CD9 expression was enhanced by adenovirus transfection,and changes in F-actin polarization,pseudopod formation and collective directional migration of epidermal cell monolayers were observed under the conditions of electric field action to clarify the role of CD9 in electric field-induced leader cell F-actin polarization and collective directional migration of epidermal cell monolayers.3.Downstream mechanism of CD9-mediated F-actin polarization of leader cells and collective directional migration of epidermal cell monolayers under electric field conditionsFirstly,the effects of CD9 overexpression on ADAM17 activity and EGFR phosphorylation in epidermal cell monolayers under electric field conditions were clarified by enzyme-linked immunosorbent assay(ELISA)and Western Blot techniques;Secondly,overexpression of CD9,inhibition of ADAM17 activity and EGFR phosphorylation,and exogenous addition of recombinant HB-EGF were used to observe the changes of leader cell’polarization,pseudopodia formation and collective directed migration under the electric field;Finally,normal epidermal cells were co-cultured with CD9 overexpressing cells to observe the changes of polarization,pseudopod formation in leader cells and collective directional migration in co-cultured epidermal cell monolayers with different CD9 expression levels.The effects of exogenous addition of anti-HB-EGF on leader cells’ polarization,pseudopod formation and collective cell directional migration in the above co-cultured epidermal cell monolayers were observed by exogenous addition of anti-HB-EGF.Results:1.Electric field induced collective migration of epidermal cell monolayers toward the anode in a field strength-dependent manner,and F-actin polarization and dynamic pseudopods along the migration direction were formed in leader cells at the monolayers;and inhibition of F-actin polarization completely eliminated electric field-induced leader cell’ pseudopod formation and collective directional migration.2.Confocal microscopic observation showed that CD9 and F-actin co-localized in epidermal cells,and electric field significantly down-regulated CD9 expression in leader cells of epidermal monolayers;CD9 overexpression eliminated electric field-induced F-actin polarization in leading cells and collective directional migration in epidermal monolayers.3.Electric field significantly induced increased activity of ADAM17 and EGFR in epidermal cell monolayers,while the enhanced activity of both could be suppressed by CD9 overexpression.4.Overexpression of CD9,inhibition of ADAM17 activity and EGFR phosphorylation all inhibited electric field-induced F-actin polarization,pseudopod formation and collective directional migration of epidermal monolayer leaders,while exogenous addition of recombinant HB-EGF can reverse the inhibition of CD9 overexpression and ADAM17 activity inhibition on F-actin polarization,pseudopodia formation and collective directed migration in the epidermal monollayer.5.In epidermal cell monolayers co-cultured with normal cells and CD9 overexpressing cells,pseudopods of CD9 overexpressing cells on the anodic side of the electric field and collective directional migration of cell monolayers were effectively restored,and this restoration could be completely inhibited by exogenous addition of HB-EGF neutralizing antibody.Conclusions:Our results suggest that electric fields induce epidermal monolayer directed collective migration by downregulating CD9 expression in leader cells.This downregulation of CD9 expression mediates F-actin polarity distribution and pseudopod formation in leader cells by promoting ADAM17/HB-EGF/EGFR signaling activation,while the released HB-EGF may also coordinate the polarization of neighboring leader cells through its paracrine effect.Our study not only provides new ideas on the molecular mechanism of electric field-induced epidermal monolayer directed collective migration,but also may provide potential therapeutic targets for the treatment of acute or chronic wounds.