The Regulatory Effects And Mechanisms Of P120-Catenin In Organ Injury

Part 1 The regulating effects and mechanisms of p120 in ventilator induced lung injuryExperimentl The effects and mechanisms of cyclic stretch on pulmonary epithelial cellsBackgroundMechanical ventilation has been the important method for patients with infection, shock, trauma, especially the acute respiratory distress syndrome (ARDS), which will lead oxygenation dysfunction. However, experience from clinic and experiments suggested that the ill would become worse and the oxygenation dysfunction would be aggravated during mechanical ventilation, which was defined as ventilator-induced lung injury (VILI). The morbidity and mortality of patients with VILI increased yearly.Therefore, it will be significant beneficial for clinic and social to explore the mechanism of VILI.VILI is characterized with pulmonary edema, high permeability of pulmonary capillary.So, it will be great of significance to maintain the integrity of pulmonary membrane and the permeability of pulmonary vascular.The direct index that responses to the integrity of pulmonary membrane and the pulmonary permeability is the cell-cell junctions.Cell-cell junctions are mainly composed of adherin junctions and tight junctions. Adherin junction proteins include p120, E-cadherin, α-catenin, β-catenin; Tight junction proteins are mainly occludin and ZO-1. Mechanical stretch could destroy the cell-cell junctions,increase cell gap and induce pulmonary edema. However, the mechanisms of mechanical stretch induced lung injury are not completely elucidated. Therefore, the key problem is to explore the mechanisms of mechanical stretch on cell junctions.Studies have proved that stimulations such as inflammation, hypoxia could active tyrosine-protein kinase c-Src and induced inflammatory response. c-Src is a member of Src family and participates in the cell signaling in cells. Previously study showed that p120 was a substrate of c-Src and c-Src activation could degradep120. However, the effects of c-Src on the cell junctions in mechanical stretch induced lung injury are not clear, especially the effects on tight junction proteins. And the mechanisms need further exploration.ObjectivesThis project imitatedVILI in clinic through stretching pulmonary epithelial cells with different stretch models to explore:(1) The influence of mechanical stretch on the expression and distribution of junction proteins of pulmonary epithelial cells. (2) To explore the mechanisms of mechanical stretch induced cell junction dysfunction by using c-Src inhibitor.Methods1. This project stretched pulmonary epithelial cells (MLE-12) using FX-4000T Flexercell instrument. The stretch parameters were 20% cyclic stretch, 0.5HZ,2/4h. The indexes need be detected were as follows:(1) Western blot was used to detect the influence of mechanical stretch on junction proteins:p120, E-cadherin, occludin, a-catenin, (3-catenin.(2) Extracted the membrane proteins and cytoplasm proteins. western blot was used to detect the expression of E-cadherin in membrane and cytoplasm.(3)CO-Immunoprecipitation (CO-IP) was used to detect the binding of junction proteins:p120 and E-cadherin, occludin and ZO-1.(4) The binding of p120 and E-cadherin as well as the gap formation were observed by using immunofluorescence.2. c-Src inhibitor PP2 (100nM was dissolved in DMSO 30μl/ml) was used to treat the cells for 30 min. After 20% cyclic stretch for 2 h, the indexes were detected:(1) The activity of c-Src and p-c-Src were determined using western blot.(2) The level of p120 and occludin was determined using western blot.Results1.The influence of mechanical stretch on the cell junction and junction proteins of pulmonary epithelial cells:(1) The effects of mechanical stretch on junction proteins of pulmonary epithelial cells. The levels of p120, E-cadherin, occludin, a-catenin were decreased after 20% cyclic stretch for 2 h. After 4 h stretch, the level of these junction proteins decreased obviously except β-catenin. The results showed that β-catenin increased after cyclic stretch. And the other proteins decreased as the stretch time increased (p< 0.05).(2)The effect of mechanical stretch on E-cadherin distribution.The membrane and cytoplasm proteins were extracted after cyclic stretch for 2/4 h.The level of E-cadherin in membrane was decreased while increased in cytoplasm after 2h cyclic stretch. And this effect was aggravated after.4h cyclic stretch (p<0.05).(3) The effects of mechanical stretch on the binding of junction proteins of pulmonary epithelial cells:The binding between p120 and E-cadherin, occludin and ZO-1 decreased after 20% cyclic stretch for 2 h, and these bindings decreased obviously after 4 h cyclic stretch (p<0.05). Consistent with CO-IP results, immunofluorescenceexperiment showed similar results.(4) The effects of mechanical stretch on epithelial gap formation:The epithelial gap formation increased after 2 h cyclic stretch and the junction became worse after 4 h stretch (p<0.05).2. Mechanical stretch actived c-Src, then caused the degradation of p120 and occludin.(1) c-Src was activated after 2 h cyclic stretch, and the expression of p-c-Src increased. However, PP2 could decrease c-Src activity.(2) PP2 could restore the degradation of p120 and occludin caused by mechanical stretch.ConclusionsThis study verified:(1) Mechanical stretch could degrade the junction proteins:p120, E-cadherin, occludin, a-catenin.(2) Mechanical stretch could induce E-cadherin endocytosis.(3) Mechanical stretch could disassociate the junction proteins and increased gap formation.(4) c-Src activation was the main reason of the dysfunction of cell junctions and the epithelial gap formation.(5) c-Src inhibitor PP2 could inhibit epithelial junction dysfuntion and gap formation caused by mechanical stretch.In a summary, mechanical stretch could active c-Srckinase, which induces the degradation and disassociation of epithelial junction proteins, increase the gap formation and pulmonary membrane permeability. And these changes finally contributed to pulmonary edema. However, c-Src inhibitor PP2 could inhibit these effects induced by mechanical stretch.Experiment 2 The regulating effects and mechanisms of p120 in ventilator induced lung injuryBackgroundMechanical ventilation is an indispensable therapy for patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, irregular mechanical stresses and strains can easily cause airway and alveolar damage, known as ventilator-induced lung injury (VILI). The primary reasons of the increased alveolar membrane permeability are the destroyed integrity of alveolar membrane and the decreased expression of junction proteins. Pulmonary epithelial cells will undergo biaxial stretch as the surface area of the basement membrane increases which can affect the integrity of alveolar membrane during mechanical ventilation. Mechanical stretch in the form of cyclic stretch induces structural and cytosolic changes in alveolar epithelial cells and finally causes alveolar epithelial barrier dysfunction and pulmonary hyper permeability which are similar with previously studies. Although loss of integrity of intercellular junctions caused by the decreased junction proteins is a major factor of increased epithelial permeability, the mechanisms of this effect have not been completely elucidated.Adherens junctions and tight junctions are two important components to keep the integrity of epithelial barrier. Adheren junctions are depending on the association of p120, E-cadherin, β-catenin and a-catenin. They have been shown to act as transducers, mediating the transduction of shear-stress signals into vascular endothelial cells.Some studies have revealed that mechanical stretch could active c-Src and caused the degradation of p120 and occludin. p120 distributes in vascular endothelial cells, pulmonary epithelial cells, fibroblasts in vertebrates. Previous studies showed that p120 was associated with the tumor cell migration and diffusion. And p120 in human lung endothelial cells was related with inflammation. p120 degradation could enhance inflammation. p120 was adherin junction protein but occludin was also decreased as the activation of c-Src during mechanical stretch. Therefore, p120 degradation induced by mechanical stretch might affect the expression and distribution of other junction proteins of epithelial cells. And the mechanism which is important for VILI needs further exploration.ObjectiveCyclic stretch on pulmonary epithelial cells and mice VILI model were used to explore:(1) To explore the regulatory effects of p120 on RhoA activity and tight junction.(2) To explore the effects and mechanisms of p120 in VILI.Methods1. MLE-12 cells were treated with c-Src inhibitor PP2 and RhoA inhibitor Y27632, and the cells were stretched with FX-4000T Flexercell machine for 2 h: 20% cyclic stretch,0.5 HZ. The activity of RhoA kinase, the expression of E-cadherin, occludin and cell gap formation were detected by pull down assay, western blot and immunofluorescence.2. MLE-12 cells were transfected with p120 siRNA/p120 cDNA and then the cells were treated with 20% cyclic stretch for 2 h.(1) Pull down assay was used to detect RhoA activity.(2) Extracted the membrane and cytoplasm protein. The distribution of E-cadherin was determined by western blot.(3) Immunoprecipitation was used to determine the binding of p120 and E-cadherin, occludin and ZO-1.(4)Immunofluorescence would show the connection of p120 with E-cadherin and the gap formation.3. C57BL/6 mice were injected with p120 siRNA-liposome mixture through retinal vein plexus to deplete most p120 in mice. Mice were randomly assigned into four groups (n=5):Control group,mechanical ventilation group, p120 knock out group(KO), p120KO and mechanical ventilation group. Some other mice were pretreated with PP2 and Y27632 through intraperitoneal injection. After mechanical ventilation, the indexes which were detected were as follows:(1) The p120 knock out efficiency was determined by western blot.(2) Pull down assay was used to detect RhoA activity.(3) The expression of E-cadherin and occludin was examined by western blot.(4)Enzyme-linked immuno sorbent assay (ELISA) was used to detect the production of IL-6, IL-8, TNF-a.(5) Lung tissue was obtained to determine Wet/Dry ratio.Results1. The activity of RhoA increased in cyclic stretch group. The degradation of E-cadherin,occludin and the gap formation of MLE-12 increased in cyclic stretch group. All of these results caused by mechanical stretch wasreversed in PP2 or Y27632 treatment group.2. (1) Mechanical stretch could active RhoA kinase. And RhoA activity increased in p120 siRNA group; also it was enhanced after mechanical stretch in p120 siRNA group.(2) E-cadherin degradation and endocytosis were increased in p120 siRNA group and was enhanced after cyclic stretch (p<0.05). However, p120 overexpression could reverse E-cadherin degradation and endocytosis.(3) CO-IP results showed that the expression of occludin and its association with ZO-1 were decreased in p120 siRNA group, which were enhanced after mechanical stretch. However, the degradation of occludin and its disassociation with ZO-1 were restored in p120 overexpression group. And the binding between E-cadherin and occludin increased in p120 overexpression group.(4) Immunofluorescence suggested that the binding between E-cadherin and occludin decreased, the epithelial cell gap increased in p120 siRNA group and p120 siRNA treatment with mechanical stretch group while restored in p120 overexpression group.3. (1) 70% of p120 was depleted by p120 siRNA-liposome mixture through retinal plexus injection.(2) Mechanical ventilation could active RhoA kinase. And RhoA activity increased in p120 KO group; also it was enhanced after mechanical ventilation in p120 KO group.(3) The expression of E-cadherin and occludin decreased after ventilation in lung tissue. However, PP2 or Y27632 could restore the junction proteins’ degradation (p<0.05). Compared with control group, the expression of E-cadherin and occludin decreased, and it decreased obviously after mechanical ventilation in p120 knock out group (p<0.05)(4) The production of IL-6, TNF-ain bronchoalveolar lavage fluid (BALF) increased in mechanical ventilation group while reduced in PP2 or Y27632 treatment group (p<0.05). And the cytokines increased obviously in p120 KO group with or without mechanical ventilation (p<0.05)(5) Lung tissue were obtained and the W/D increased in mechanical ventilation group while reduced in PP2 or Y27632 treatment group (p<0.05). And the W/D increased obviously in p120 KO group with or without mechanical ventilation (p<0.05)ConclusionsThis study concluded that:1. PP2 or Y27632 could restore the degradation of junction proteins and epithelial gap formation caused by mechanical stretch.2. p120 degradation could active RhoA kinase.3. E-cadherin endocytosis, occludin degradation, the disassociation of occludin and ZO-1, epithelial gap formation were all increased in p120 knock down group while restored in p120 overexpression group.4. High volume ventilation could cause the degradation of cell junction proteins, the production of IL-6, TNF-a,and increase W/D, which lead to pulmonary edema. However, PP2 and Y27632 could restore the damage caused by mechanical ventilation.5. The degradation of lung junction proteins, the production of IL-6 and TNF-a and the W/D ratio were all increased in p120 knock out group, which aggregated VILI.Therefore, this project pointed that c-Src activation caused by mechanical ventilation could induce p120 degradation, which in turn enhanced adherin junction dysfunction and tight junction dysfunction through increasing E-cadherin endocytosis and RhoA activation-induced occludin degradation. p120 was an important media between adherin junction and tight junction, which was the new target of the treatment of VILI.Part 2 The regulatory role of p120 in cardiac dysfunction in sepsisBackgroundSepsis remains a major cause of death among hospitalized patients. The incidence of sepsis is still high and with an estimated 18 million new cases yearly worldwide. The early death during sepsis is mainly ascribed to the distributive shock or the cardiogenic form of septic shock. Without intervention, severe and systematic inflammatory responses develop quickly along the course of sepsis, causing various tissue damage and organ dysfunction, eventually leading to organ failure and death. Cardiac dysfunction is one of the leading causes of mortality in sepsis due to its implication of cardiac contractile dysfunction and circulation disorders. The mechanisms of cardiac contractile inhibition in sepsis are complex and poorly defined.Myocardium contraction is related with the intercalated discs. The integrity of the cardiomyocytes intercalated discs, which is composed of desmosomes, adherens junctions (AJs) and gap junction, plays an essential role in maintaining the normal myocardial contraction. This was proved by the fact that deficiency of AJs could destroy the intercalated discs and lead to cardiomyopathies.The cardiomyocytes AJs are mainly composed of p120, N-cadherin and β-catenin. Dephosphorylation of p120 has been shown to induce AJs dysfunction in endothelial cells, and cardiomyocytes. However, some other studies suggested that Anx5 could bind to the phosphatidylserine on the cell membrane in a calcium-dependent manner.Therefore, we concluded that Anx5 might exert its protective effect on cardiomyocytes during sepsis by modulating the function of AJs via p120. This was verified by Anx5, PKCainhibitor and PKCasiRNA. p120 played an important role on cardiomyocyte contraction in sepsis.ObjectsThis study used the LPS treated cardiomyocytes and H9c2 cells to explore the regulatory role of p120 on myocardium contraction in sepsis.Methods1. Mice cardiomyocytes were treated with LPS (1μg/ml) for 0、2、4、6h, and the following indexes were determined:(1) p120 expression, PKCαwere detected with western blot. (2) The dephosphorylation of p120 and the binding between p120 and N-cadherin were determined by CO-IP.2. H9c2 cells or cardiomyocyte cells were treated with PKCainhibitor G66976 or PKC a siRNA, after LPS (1μg/ml) treatment for 0,2、4h, the following indexes were detected:(1) p120 expression, PKCawere detected with western blot. (2) The dephosphorylation of p120 and the binding between p120 and N-cadherin were determined by CO-IP. (3) The relation between p120 and N-cadherin was confirmed by immunoflourescence.Results1. Compared with control group, p120 level decreased, p120 dephosphorylation increased, PKCaactivity increased, the binding between p120 and N-cadherin reduced in LPS treatment group.2. H9c2 cells were treated with PKCainhibitor or PKCasiRNA. After LPS challenge, p120 dephosphorylation increased, PKCaactivity increased, the binding between p120 and N-cadherin reduced. However, PKCainhibitor Go6976, PKCasiRNA or Anx5 all could restore this effects induced by LPS.Conclusions1. LPS could induce p120 dephosphorylation, p120 degradation and the disassociation with N-cadherin.2. PKCainhibitor G66976, PKCasiRNA or Anx5 all could restore the effects induced by LPS including p120 dgradation, p120 dephosphorylation and the disassociation with N-cadherin as well as the cardiomyocytes gap formation.