Role Of Integrin-Erk1/2 Signaling Pathway In Oral Epithelial Adhesion And Rete Peg Elongation And Its Mechanism

Part 1 KGF enhances oral epithelial adhesion and rete peg elongation via integrins Objective:Oral epithelial adhesion to the lamina propria underlies the physiologic function of the oral mucosa and contributes to resisting bacterial invasion,preventing body fluid loss,and maintaining routine chewing;thus,understanding the factors that positively influence oral epithelial adhesion is a research topic of great interest.Rete pegs contribute to oral epithelial adhesion by enlarging the contact areas,whereas integrins are the major molecules that mediate epithelial cell adhesion to the basement membrane.Keratinocyte growth factor(KGF)can promote both rete peg elongation in the skin and the expression of integrins in various cell types.Herein,we tested the effects of submucosal injection of KGF in the ventral surfaces of rat tongues on oral epithelial adhesion.Methods:Age-and weight-matched healthy male SD rats were submucously injected with PBS,KGF(400ng/ml),HYD-1(4mg/ml),and KGF+ HYD-1(30?L,n=8)every other day.The length of rete peg,the expression of integrin subunit ?6,?4,?3,and ?1,and the adhesion strength of the oral epithelium were assessed.The impact of KGF on the expression of integrin subunit ?6,?4,?3,and ?1 was detected by real-time RT-PCR,Western blot,and immunocytochemistry.In vitro adhesion assay in HIOEC cells after blocking the integrin was performed.The expression of integrin in human oral mucosae of different areas was detected and the correlation of integrin expression with length of rete peg was analyzed.Reulsts:Topical injection of KGF elevated the adhesive forces,elongated the rete pegs,and increased the abundance of integrins,KGF,and KGF receptor on the rat tongue ventral surface.However,HYD-1(Lys-Ile-Lys-Met-Val-Ile-Ser-Trp-Lys-Gly),an integrin antagonist,inhibited the KGF enhanced epithelial adhesion and rete peg elongation.Moreover,KGF promoted the expression of integrin subunits ?6,?4,?3,and ?1 in human immortalized oral epithelial cells in 2-and 3-dimensional culture systems.In vitro cell attachment assays demonstrated that HYD-1 inhibited the adhesion of human immortalized oral epithelial cells on Matrigel.Strikingly,the expression of integrins,KGF,and KGFR in human masticatory mucosae with longer rete pegs was more abundant than that in the lining mucosae with shorter rete pegs.In addition,rete peg lengths were positively correlated with the expression levels of integrins,KGF,and KGFR.Conclusions:These findings indicate that KGF strengthens oral epithelial adhesion and rete peg elongation via integrins.Part 2 KGF induce podosome formation in HIOECsObjective:KGF enhances oral epithelial adhesion and rete peg elongation via integrins.However,rete peg elongation underlines the proliferation and migration of epithelial cells,as well as remodeling of basement membrane,which may compromise the epithelial adhesion.Podosomes are cell-matrix contact areas that combine several abilities including adhesion and matrix degradation.This study aims to explore the possibility of podosome formation in human immortalized oral epithelial cells(HIOECs).Methods:The podosome-like structures were identified by co-localization of F-actin and cortactin with laser scanning confocal microscopy.In addition,the key component of podosome such as MMP14 and integrins were identified by laser scanning confocal microscopy.The impact of KGF on the expression of MMP family including MMP2,MMP9,and MWP14 was detected by real-time RT-PCR and Western blot.In situ matrix degradation assay was perfomed to demonstate the ability of podosome-like structures to degrade matrix.Results:Our results demonstrated that KGF promoted the formation of podosome-like structures in HIOECs.KGF induced the expression of MMP2,MMP9,and MVP 14.Moreover,integrins and MMPs were found co-locolized with the podosome-like structures.Further degradation assay demonstrated that these podosome-like structures could degrade matrix.Conclusions:KGF induces podosome formation in HIOECs,indicating a novel mechanism by which integrins enhance oral epithelial adhesion and rete peg elongation.Part 3 Integrin-Erkl/2 signaling in KGF-induced podosome formation in HIOECsObjective:The above studies demonstared that KGF induced podosome formation in HIOECs.However,the associated signaling pathway remained unclear.Integrins are vital components of podosome,and play essential role in the assembly of podosome.Therefore,the aim of this part was to explore the regulatory role of integrins and its related signaling pathway in podosome formation in HIOECs after KGF treatment.Methods:The effects of integrins on KGF-induced podosome formation were investigated by HYD-1,an antagonist for integrin subunit a6,?4,?3,and ?1,under laser scanning confocal microscopy.To validate the crudial integrin subunits,the HIOECs were treated with specific blocking antibody for integrin subunit ?6,?4,?3,and ?1 To screen the key signaling pathway,the expression and phosphorylation level of MAPK,Akt,Jak2,and Stat3 upon KGF treatment were detected by Western blot.To ascertain the indispendable role of Erkl/2 and Akt in KGF-induced podosome formation,we pretreated HIOECs with U0126 and Ly294002 before KGF addition.The impact of Erkl/2 and Akt on HIOECs were analyzed by MTT and EdU assays.The phosphorylation levels of Erkl/2 and Akt were detected by Western blot after the knockdown of integrins with their specific siRNA.Results:HYD-1 significantly inhibited the KGF-induced podosome formation.Both inhibition of integrin subunits ?1 and ?4 with their specific blocking antibodies 12G10 and 3E1 abrogated KGF-induced podosome formation.KGF promoted the activation of Erkl/2 and Akt.Inhibition both of Erkl/2 with U0126 and Akt with Ly294002 could upregulated the expression of integrin subunits ?1 and ?4.However,only U0126 abrogated KGF-induced podosome formation in HIOECs.Knockdown of integrin subunits ?1 and ?4 with specific siRNA downregulated the phosphorylation levels of Erkl/2 and Akt.Conclusions:These results demonstrated that integrins-Erkl/2 signaling play essential role in KGF-induced podosome formation in HIOECs.Both integrin ?4 and ?1 are indispensable.