Effect Of P120 On NF-κB Signaling Pathway During The Airway Epithelial Inflammation Induced By Mechanical Scratching

BackgroundAirway consisting of trachea and bronchi is the first defense to resist harmfulstimuli. Mechanical scratching as the main stimulus in vitro model maycause airwayinjury, resulting in polarization, necrosis and secretion of inflammatory cytokines ofthe bronchial epithelial cells. This process repeating persistently can develop intochronic airway inflammation, which is the basis of various lung diseases, such asCOPD,chronic pulmonary fibrosis and lung cancer. Numerous studies indicate thatthe activation of nuclear factor-κB(NF-κB) signaling pathway is involved in the cellgrowth,proliferation,apoptosisandinflammatorygenetranscription,etc.Itisthecoreof inflammatory responses, and closely associated with mechanical scratchinginducedairwayinflammation.Howevertheexactmechanismisstillunknown.Inhibitor of NF-κB (IκB), a master switch for NF-κB activating, usually bindsNF-κBdimmerintoNF-κB/ IκBcomplexinrestingcells,andthiscytoplasmicNF-κBis inactive. With external stimuli such as mechanical scratching inducing theactivation of IκB, NF-κB immediatelybecomesreleased and activated. It translocatesinto nucleus and binds with target genes, for example IL-8, thus enhancinginflammatory gene transcription. p120-catenin ( p120 ), belongs to a new member ofthe Armadillo protein family, which is identified when screening a substrate foroncogenicSrc familytyrosinekinase.Previous studies focusedonp120-mediatedcelladhesion and tumorgenesis. Recent studies revealed that p120 is associated with skin,salivary glands inflammation and inflammatory bowel diseases through invoking theNF-κBsignalingpathway. Buttheresearchontherelationshipofp120andtheairway inflammation is rare. Therefore our studies focused on changes of p120 and its effectson NF-κB signaling pathway during the airway inflammation induced by mechanicalscratching.ObjectiveUsing a widely used in vitro model by scratching human bronchial epithelialcells(HBECs), we investigate the expression changes of p120 and its effects on NF-κB signaling pathway. This study may bring theoretical and experimental evidences forfurther exploring the mechanism of airway inflammation.MethodsIn the model of airway inflammation by scratching a monolayer of HBECs invitro, we utilized Western blot to detect the expressions of p120, NF-κB p65 and IκBα.Next, we examined NF-κB p65 translocation into nucleus by isolation of cytoplasmicand nuclear proteins. Finally, we detected the expression of IL-8 by enzyme-linkedimmunosorbent assay(ELISA).ResultsBy Western blot, we examined the expression of p120 isoforms in HBECs,mainly composed of isoform 1(120KD) and isoform 3(100KD). And the expressionof isoform 1 was weak. Then we found that scratching decreased p120 and IκBα, butsignificantly increased the amount of NF-κB p65 at different time points. It indicatesthat the phosphorylation and degradation of IκBαactivated NF-κB. Moreover, thedecreasing of p120 activated NF-κB signaling pathway. Isolation of cytoplasmic andnuclear proteins showed NF-κB p65 translocation into nucleus. Meanwhile, thenuclear expression of NF-κB p65 was gradually increased in a time-dependentmanner after scratching. Finally ELISA detected the increased production of IL-8(p﹤0.05).Conclusions1 Mechanical scratching inhibits the expression of p120 in HBECs, and activatesNF-κB signaling pathway.2 Phosphorylated degradation of IκBαinvolves in the activation of NF-κB signaling pathway after mechanical scratching HBECs.3After activation of NF-κB signaling pathway by scratching, p65 nucleartranslocation promotes the production of IL-8, and invokes the inflammatoryresponses.Summaryp120 may inhibit the airway inflammation through a negative regulation onNF-κB signaling pathway in themechanical scratching induced airway inflammatoryresponses.