The Effects And Mechanisms Of Keratinocyte Migration Regulated By CD9/ ADAM17

Background and purpose:Re-epithelialization of wounds involves several keratinocyte functions: proliferation,migration,and differentiation.The key step is keratinocyte migration,an essential aspect for understanding chronic non-healing wounds.According to our previous study,wound repair was delayed in CD9-knockout mice,indicating that CD9 played a key role in wound healing.CD9 down-regulation is beneficial to keratinocyte migration.CD9 is a highly conserved and intact transmembrane protein composed by two extracellular loops and short intracellular ends as well as four transmembrane domains;it plays an important role in cell migration,motility and adhesion.By contrast with many other cell surface proteins,CD9 does not have an obvious receptor function,CD9 may participates in the organization of surface multiprotein complexes through association with other transmembrane molecules,including integrins,ADAMs and signaling receptors,thereby mediating various cellular and physiological processes.However,the regulation mechanisms of interaction between CD9 and other transmembrane molecules in keratinocyte migration are poorly understood.Our previous study found that CD9 down-regulation triggered the switch from ?v?5 to ?v?6 integrin in keratinocytes,a critical step for cell migration;however,the functional regions mediating CD9 and integrin ?v?5 interaction in keratinocyte migration remains unclear Moreover,CD9 complexes with integrins including ?2?1 or ?3?1 do not participate in keratinocyte migration.Therefore,CD9 may regulate keratinocyte migration by activating other non-integrins transmembrane molecules.A disintegrin and metalloproteinases(ADAMs)are a family of transmembrane proteinases which take charge of proteolytic cleavage as well as release of various substrates from the cell surface,which greatly impact the wound healing,and tumorigenesis.The ADAM family consists of 22 known members;however,only ADAM17 is thought to be a key regulator in repair of skin.ADAM17-/-mice are short of various kinds of EGFR ligands,and,due to the decreases of HB-EGF and TGF-?,suffer many serious failures with regard to the epithelial morphogenesis and maturation.In ADAM17 active epidermis,EGF family molecules such as HB-EGF,TGF-? and amphiregulin(AREG)are released,and EGFR activates enhancement.It is known that EGFR ligands promote the migration of keratinocyte,thereby significantly improve wound healing,especially in the early reaction to wounding.In particular,HB-EGF and TGF-? are responsible for keratinocyte migration during wound healing,meanwhile the HB-EGF contributes to the re-epithelialization and accelerates wound healing.The data showed that ADAM17 played a key role in keratinocyte migration and wound re-epithelialization.Nevertheless,the mechanisms of ADAM17 maturation or activation have not been elucidated in keratinocytes.According to a study conducted recently using the super-resolution microscopy,it seemed that a majority of tetraspanin nanoclusters contain ADAMs.This is beneficial for explaining the specific function of tetraspanin CD9 with transmembrane metalloproteinases.Here,we described investigations of the interactions between CD9 and ADAM17 in keratinocyte migration.We demonstrated that the sheddase activity of ADAM17 was activated by CD9 down-regulation,increasing cleavage and release of HB-EGF,as well as activation of the EGFR/ERK signaling pathway,promoting keratinocyte migration and wound healing.The purpose of this study was to clarify that the sheddase activity of ADAM17 was regulated by CD9,increasing cleavage and release of EGF,which is beneficial for keratinocyte migration and wound healing.It is a good theoretical mechanism for deeply understanding chronic non-healing wounds.Contents and methods:1.The co-localization and relationship of CD9 and ADAM17The expression and localization of CD9 and ADAM17 in the skin epidermis of C57 mice,primary cultured mouse epidermal cells and HaCaT(human epidermal cell line)cells were observed by immunofluorescence double staining and laser confocal observation,and the interaction between CD9 and ADAM17 was further clarified by immunoprecipitation2.Effect of CD9 on expression and function of ADAM17 and its relationship with keratinocyte migrationTo assess the role of CD9 in ADAM17 maturation and activation in keratinocytes,we constructed recombinant lentivirus vectors to silence CD9(CD9-shRNA)and overexpress CD9(Ad-CD9)by infecting HaCaT cells and C57-MKs,and then carried out the Western blotting on CD9 and ADAM17.The enzyme activity of ADAM17 was assessed using a TACE protease activity kit.A cell scratch wound assay was used to evaluate keratinocyte migration taking the ADAM17 inhibitor TAPI-2 and siADAM17 as siRNA-mediated knockdown of ADAM17.3.The downstream signaling participate in keratinocyte migration was regulated by CD9/ADAM17In the foregoing scratch assay,ADAM17's substrates were examined in term of shedding,including amphiregulin(AREG),HB-EGF and TGF-?,during the period that cells were cultured without exogenous EGF.Representative Western blot results showing the effect of recombinant on phosphorylation of EGFR/ERK in CD9-silenced keratinocytes treated with siADAM174.Identification of functional domain for interaction between CD9 and ADAM17 in keratinocyte migrationThe interaction domain between metalloproteinase ADAM17 and CD9 was identified by membrane yeast two-hybrid system(ubiquitination system).On this basis,mutual domain mutant plasmids were constructed,transfected with epidermal cells,and immunoprecipitation was performed to detect their interactions,and ADAM17 activity was analyzed,as well as its influence on epidermal migration and movement.Results:1.CD9 and ADAM17 were expressed both on the cell surface and in the cytoplasm,co-localization of ADAM17 and CD9 was particularly evident on the cell surface.CD9 immunoprecipitates of both cell types saw bands which correspond to the mature form of ADAM17,suggesting that these two proteins exhibited a cross-linking on the cell surface.2.Compared with the vector group,CD9-silenced keratinocytes caused significant increases of ADAM17 sheddase activity,58% in HaCaT cells,and 67% in C57-MKs.Compared with the mock group,CD9-overexpression in keratinocytes caused a significant decrease of ADAM17 sheddase activity,decreasing 22.6% in HaCaT cells,and 35% in C57-MKs.The improvement in cell motility by CD9-silenced was suppressed by TAPI-2 treatment,and was abolished by siADAM17 transfection;after TAPI-2 treatment,area of wound closure was reduced 27% in CD9-silenced HaCat cells,and 25% in CD9-silenced C57-MKs.3.AREG and HB-EGF exhibited an obvious higher shedding in CD9-silenced keratinocytes than in controls.The elevated shedding significantly reduced by ADAM17 inhibitors-TAPI-2 in CD9-silenced keratinocytes.Anti-HB-EGF displayed stronger inhibition of keratinocyte migration,area of wound closure was reduced 27.4% in CD9-silenced HaCat cells and 21% in CD9-silenced C57-MKs.The cell motility assay showed that neutralizing anti-HB-EGF mAbs significantly suppressed the motility speed of CD9-silenced keratinocytes.4.Down-regulation of CD9 increased phosphorylation of EGFR,ERK and JNK in keratinocytes,this was prevented by si-ADAM17 transfection.However,adding the recombinant HB-EGF to cultures treated with si-ADAM17 of CD9-silenced keratinocytes greatly stimulated the EGFR/ERK phosphorylation.5.The functional verification group showed normal colony growth on the DDO plate,indicating that the co-transformation experiment was successful,and normal growth on the DDO/X plate was shown in blue,indicating that pbt3-n-bait interacts with post1-nubal protein and activates the reporter gene expressionConclusion:In conclusion,our findings demonstrated that down-regulation of CD9 activated the ADAM17 sheddase activity in keratinocytes,which is critical for cell migration and EGFR ligands shedding.Moreover,CD9 associated with ADAM17(i.e.catalytically active)on the surface of keratinocytes,which negatively regulated sheddase activity of ADAM17.Importantly,CD9/ADAM17 axis played a key role in keratinocyte migration via activation of EGFR/ERK signaling pathway.On that account,activating the pathway is likely to be an attractive target to improve the acute and chronic cutaneous wound healing.