Effects Of Different Phosphorylation Sites Of Cortactin On Airway Mucus Secretion Under Simulated Airway Shear Stress

Objective:To investigate the effects of different key phosphorylation sites of Cortactin on the secretion of MUC5AC under the effect of simulating optimal airway shear stress.Methods:?1?Construction of the wild-type CTTN pTSB plasmid.The main phosphorylation sites?Y421,Y470,Y486?of CTTN gene were mutated to alanine?A?by site-directed mutagenesis to simulate the non-phosphorylation status of the related sites.The recombinant plasmids PTSB02-GFP-CTTN Y421A PTSB02-GFP-CTTN Y470A,PTSB02-GFP-CTTN Y486A,PTSB02-GFP-CTTN,were constructed.Enzyme digestion identification and sequencing.?2?The shear stress simulation device refers to the experimental study of Tarran et al.and uses a phase motion instrument to simulate the shear stress of the airway epithelium by alternating the exhalation and inhalation during human breathing through alternating acceleration/deceleration.The increased shear stress and respiratory rate under pathological conditions are simulated by adjusting the time and frequency of acceleration/deceleration.In order to simulate the shear stress effect of airflowonairwayepithelialcellsunderpathologicalconditions,the acceleration/deceleration parameters were set to 1.2 s/times,30 times/min for 35 min.?3?Human bronchial epithelial cells?16HBE?were cultured in a 5%CO₂ incubator at37?.Cells in good condition were seeded in 6-well plates at 5×105 cells/well and placed in a 37?,5%CO₂ incubator.Medium culture,when 16HBE cells grow to a confluency of about 70%,they are rando mly divided into 6 groups:group?A?Y421A,group?B?Y470A,group?C?Y486A,group?D?wild type CTTN,group?E Blank plasmid negative control,group?F?blank control.16HBE cells were transfected with lipo3000 reagent.After 24 hours of incubation,shear stress stimulation was applied,and the cells were analyzed by transferring them back to the incubator for 24 hours and then analyzing the transfected cells.?4?The relative levels of Cortactin and p-Cortactin Y421?p-Cortactin Y470?p-Cortactin Y486 were detected by Western Blotting.The mRNA levels were detected by RT-PCR.The relative secretion of extracellular MUC5AC and the content of intracellular MUC5AC were determined by enzyme-linked immunosorbent assay?ELISA?.Results:?1?By gene sequencing and restriction enzyme digestion;the CTTN gene major phosphorylation sites?Y421,Y470,Y486?were successfully site-directed,recombinant plasmids?PTSB02-GFP-CTTN-Y421A,PTSB02-GFP-CTTN-Y470A,PTSB02-GFP-CTTNY486A?,PTSB02-GFP-CTTN?was successfully constructed and can be used in subsequent experiments.?2?Transfection by Lipo3000 reagent,fluorescence microscopy showed that the plasmid was successfully transfected,and the transfection water reached more than80%.?3?The expression of Cortactin mRNA was detected by real-time PCR.Compared with the negative control and the blank control,the levels of Cortactin mRNA were significantly increased in the cells transfected with each group.The expression of each group was compared between the transfected groups.Cortactin mRNA levels are essentially the same.?4?The extracellular secretion of MUC5AC in each group was determined by ELISA.The extracellular secretion level of MUC5AC in group A was significantly lower than that in group B,C and D?P<0.01?.The extracellular secretion level of MUC5AC in group B was lower than D.The difference was statistically significant?P<0.05?.The extracellular secretion level of MUC5AC in group A was significantly higher than that in group E and F,the difference was statistically significant?P<0.05?.There was no significant difference between group C and group D.The significance?P>0.05?,E group and F group,the difference was not statistically significant?P>0.05?.?5?We used Western Bloting to detect the relative levels of Cortactin protein and p-Cortactin Y421,p-Cortactin Y470,p-Cortactin Y486 protein in each group.Compared with E and F groups,the expression levels of Cortactin protein in group A,B,C and D were significantly increased.The relative expression levels of Cortactin were basically the same in each plasmid transfection group.Compared with group B,C and D,the relative expression of p-Cortactin Y421 protein in group A was significantly decreased.The relative content of phosphorylated Cortactin Y470 and Cortactin Y486 in each group had no significant correlation with extracellular MUC5AC secretion.Conclusion:The phosphorylation of the Cortactin phosphorylation site T421 under the simulated airway shear stress promoted the secretion of MUC5AC.The phosphorylation of T470 promoted the secretion of MUC5AC.The phosphorylation of T486 had little effect on the secretion of MUC5AC.