The Synergistic Effect Of Exocyst And Cortactin On Mucus Hypersecretion

Objective We sought to explore the intracellular location andexpression of tethering factor Exocyst complex in targeted population andin human bronchial epithelial cells (16HBE). We also sought to clarify therole of Exocyst complex in Neutrophil elastase (NE)-provoked mucushypersecretion and its potential synergistic effect with the corticalactin-binding protein Cortactin in this process.Methods(1) The expression and location of Exocyst complex in the bronchialepithelial cells from COPD patients and health persons was examined byimmunohistochemistry, real-time PCR and western blotting. The locationand expression of Exocyst complex in16HBE cells presence or absencewith NE was detected by immunofluorescence staining, real-time PCR andwestern blotting as well.(2) We constructed the knockdown of Sec3in16HBE cells by usingtwo specific shRNA against to Sec3(Sec3-shRNA1and Sec3-shRNA2).For transfection, the knockdown of Sec3was measured by real-time PCRand western blotting. The two shRNAs were established to identify the function of Sec3in NE-induced airway mucus hypersecretion. And theexpression of MUC5AC mRNA and protein was examined by real-timePCR and ELISA, respectively.(3) Transfection with Sec3-shRNA, Cortactin-shRNA separately orjointly. The levels of MUC5AC mRNA and protein were measured byreal-time PCR and western blotting. Co-immunoprecipitation andimmunofluorescent double staining were used to detect the interaction andco-location between Sec3and Cortactin to clarify their synergistic effect onNE-induced mucus hypersecretion.Results(1) The levels of Sec3protein and mRNA were significantlyincreased in patients with COPD compared with the levels seen in healthysubjects. Moreover, Sec3positive immunoreactivity was significantlydetected at the top membranes of bronchial epithelial cells which beside theluminal side. In16HBE cells, we found Sec3was transfer from cytoplasmtoward the secretory active zone of cell membrane after NE challenge.Meanwhile, NE also produced increases in Sec3mRNA and protein in16HBE cells.(2) We found that NE increased the mRNA and protein expressionsof MUC5AC in a concentration-and time-dependent manner. Toinvestigate the role of Sec3in NE-induced MUC5AC hypersecretion,shRNA experiments were performed. Then we found the shRNA1showed the perfect knockdown of Sec3. And both Sec3-shRNAs significantlyreduced NE-induced MUC5AC protein overexpression but failed toattenuate the overexpression of MUC5AC mRNA.(3) To further clarify the synergistic effect of Sec3and Cortactin,shRNAs tagteted to Sec3and Cortactin were performed separately orjointly. Cortactin-shRNA alone could significantly attenuated NE-inducedthe MUC5AC intracellular production and extracellular secretion, and thecombination of Sec3and Cortactin could completely block the extracellularsecretion induced by NE. Furthermore, the co-immunoprecipitation andimmunofluorescent double staining captured the interaction and co-locationof Sec3and Cortactin in resting state. Moreover NE could enhance theinteraction.Conclusion Sec3mRNA and protein expression in human bronchialcells is augmented in COPD patients, meanwhile it is gathered in the cellmembrane that next to the lumen of airway. NE could increase itsexpression as well, and promote it transferred from cytoplasm toward thecell membrane, indicating that Sec3might take an important function inNE-mediated airway mucus hypersecretion. In addition, we showed thatSec3-mediated mucus hypersecretion induced by NE through a synergisticeffect with Cortactin. The identification of Sec3and Cortactin-mediatedmucus hypersecretion demonstrates the new therapeutic approaches tomitigate excessive mucus accumulation in inflamed airway. However, the mechanism of Sec3and the interaction between Sec3and Cortactin inairway mucus hypersecretion awaits further experimental researches.