The Effects Of Ezrin On Mucin Hypersecretion Of Airway Epithelial Cells In Chronic Inflammatory Airway Diseases And Its Molecular Mechanisms

Objective The goals of the studies were as follows:(1) To clarify theexpression and localization of Ezrin protein in bronchial epithelium and tofurther identify the difference of Ezrin protein expression between patientswith COPD and healthy individuals.(2) To explore the expression of Ezrinin16HBE cells and its function in mucus secretion.(3) To explore theinteractions between Ezrin and moleculars involved in exocytosis ofmucin in airway epithelia, to investigate roles of the molecular signal chainof Ezrin/PIP2/MARCKS in airway mucin secretion in detail, in order todetermine the definite molecular mechanisms and potential signalingpathway of Ezrin during participating in airway hypersecretion.Methods (1) Human biopsy specimens were obtained from surgicalspecimens. The expression and localization of Ezrin in the bronchialepithelium obtained from COPD subjects and healthy individuals wereexamined by immunohistochemistry. Quantification of Ezrin immunostaining in the epithelium was performed using Image-pro Plussoftware, Version6.0. Ezrin protein and Ezrin mRNA were further examinedby real-time quantitative reverse transcription polymerase chain reaction(real-time PCR) and Western blot, respectively.(2) Recombinant plasmidsof wild-type Ezrin and point mutants of Ezrin at Thr567site(pEGFP-N1-ezrin-Wt,pEGFP-N1-ezrin-T567D,pEGFP-N1-ezrin-T567A)verified by enzyme digestion were transfected into16HBE cells and celllines expressing Ezrin T567D, T567A, Wt and pEGFP-N1labeled byEGFP which provided basis for follow-up function control were obtained.Human airway epithelia16HBE cells were cultured in vitro, and weregiven different concentrations of human neutrophil elatase (HNE).Cytotoxicity assays were performed by MTT assay to examine cellularsusceptibility to different concentrations of HNE. mRNA expressions ofMUC5AC in cells treated with HNE were measured by real-time PCR andprotein expressions of MUC5AC in supernatants and cell lysates weredetermined by ELISA, in order to select the optimal concentration of HNE.Then16HBE cells transfected with wild-type Ezrin and different mutantsof Ezrin were treated with HNE. MUC5AC protein contents in differentgroups were tested through ELISA and protein expressions ofphosphorylated Ezrin in cells transfected with different mutants wereanalysized by Western blot method, in order to make sure the effects ofdifferent mutants of Ezrin at Thr567on MUC5AC secretions and to infer a key role in mucus secretion.(3) Neomycin, a specific inhibitor of PLCwhich could inhibit the hydrolysis of PIP2and Calphostin C, a specificinhibitor of PKC were used to16HBE cells. Phosphorylated proteins ofEzrin were tested by Western blot. At the same time, the expressions andcellular locations of PIP2and Ezrin were observed throughimmunocytochemical staining and laser scanning focal microscopy in orderto define the effects of PIP2and PKC on Ezrin phosphorylation andtranslocation to the plasma memberane. Meanwhile, cultured16HBE cellstransfected with wild-type and mutants of Ezrin at Thr567sites werestimulated with HNE, phosphorylated MARCKS protein contents weredetermined by Western blot in order to detect the effect of Ezrin onphosphorylation of MARCKS. At last,16HBE cells transfected withwild-type and mutants of Ezrin at Thr567sites were stimulated with HNE,and MUC5AC secreted outside the cells were measured by ELISA in orderto reveal the effect of MARCKS on MUC5AC secretion.Results (1) Immunohistochemistry showed that the majority ofphosphorylate Ezrin immunoreactivity was localized in bronchial mucousepithelia, especially at the top of membranes of bronchial epithelial cellsrich in cilia, near the luminal side of the cytoplasm. Ezrin protein expressedas IOD/area in the bronchial epithelium was increased in lung tissue ofsubjects with COPD, with a mean (SD) of0.28(0.049) compared withthat of subjects who had no underlying chronic inflammatory airway disease, with a mean (SD) of0.19(0.036)(P <0.05). Expression of Ezrin mRNAin lung tissue of subjects with COPD was1.18±0.071fold increase whencompared with that of control group (P>0.05) and Ezrin protein of subjectswith COPD was significantly greater than those of control group (P<0.05).(2) Recombinant plasmids of Ezrin were verified by XhoI-BamH I doubleenzyme digestion, and results of enzyme digestion were identical with theprovenience, suggesting that there were target DNA fragments in theplasmid and they were reliable in the following experiments. Plasmids werealso observed via fluorescence microscope and green fluorescence wereseen in all the four groups, suggesting successful transfections. Differentconcentrations of HNE were given to16HBE cells, none of themeasurements showed significant cytotoxicity for HNE used in the presentstudies except1μm HNE. mRNA and protein expressions of MUC5AC incells stimulated with HNE increased in a dose-dependent manner, withprominent increase in0.5μM HNE group (P <0.01) and obvious elevationsin other groups (P <0.05). Hence, we chose0.5μM as the optimalconcentration for stimulation.16HBE cells transfected with differentmutants of Ezrin at Thr567site were stimulated with HNE, Western blotand immunofluorescence results showed that HNE could induce increasedphorylated Ezrin protein expression in16HBE cells transfected withwild-type Ezrin compared with the negative control (P <0.05) andtranslocations of pEzrin to plasma membrane. Compared with the vehicle control, pEzrin proteins increased obviously in cells transfected withpEGFP-N1-Ezrin-T567D (P <0.05) and mainly located at the plasmamembrane, pEzrin proteins decreased sharply in cells transfected withpEGFP-N1-Ezrin-T567A (P <0.01) without any translocation to themembrane, while pEzrin proteins increased a little in cells transfected withpEGFP-N1-Ezrin-Wt without statistical significance (P>0.05). ELISAresults showed that compared with negative control, both MUC5ACproteins secreted extracellularly and thoese stored intracellularly increasedin all groups of cells transfected with different types of Ezrin mutants afterstimulated by0.5μM HNE (P <0.05), while compared with vehicle controland wild-type Ezrin groups, MUC5AC proteins secreted extracellularly inpEGFP-N1-Ezrin-T567D group elevated significantly (P <0.01), whilethose stored intracellularly decreased obviously (P <0.05). But MUC5ACprotein secreted extracellularly in pEGFP-N1-Ezrin-T567A decreasedsharply (P <0.01) and MUC5AC stored intracellularly increased obviously(P <0.05) when compared with that in the vehicle control group.(3)16HBE cells pretreated with Calphostin C were treated with HNE and weregiven Western blot, the results showed that phosphorylated Ezrin at Thr567site and phosphorylated MARCKS decreased obviously in cells treatedwith Calphostin C--the specific inhibitor for PKC (P <0.05) whencompared with the HNE group, but didn't decreased significantly comparedwith the control group (P>0.05). Neomycin—the specific inhibitor for PLC could inhibit the hydrolysis of PIP2induced by HNE, so PIP2in theplasma membrane increased. But immunoinfluresence and laser confocalmicroscope showed that phosphorylated Ezrin protein and pEzrin located atthe membrane dicreased compared with the HNE group.16HBE cellstransfected with wild-type and mutants at Thr567of Ezrin were stimulatedby HNE, then Western blot results showed that compared with the vehiclecontrol and wild-type Ezrin groups, pMARCKS protein in cells transfectedwith pEGFP-N1-Ezrin-T567D increased significantly (P <0.05), while thatin cells transfected with pEGFP-N1-Ezrin-T567A decreased but thedifference was not statistically significant (P>0.05).16HBE cells weretransfected with pCDNA3.0vector, PSD-mutant-MARCKS plasmid andwild-type MARCKS plasmid and treated with HNE. ELISA results showedthat MUC5AC secretion reduced significantly in cells transfected withPSD-mutant-MARCKS when compared with the cell transfected withwild-type MARCKS plasmid (P <0.05).