1.Effect of phosphorylation of cortactin at different sites on secretion by airway mucus 5AC.
Qi LI ; Xiangdong ZHOU ; Man ZENG ; Youqing ZHONG ; Victor P KOLOSOV ; Juliy M PERELMAN
Journal of Central South University(Medical Sciences) 2019;44(1):1-8
To explore the role of cortical actin-binding protein (cortactin) in shear stress-induced mucin (MUC) 5AC secretion in human airway epithelial cells and the effect of phosphorylation of cortactin at different sites.
Methods: HBE16 airway epithelial cells were cultured, and then transfected with mutation carrier, such as pEGFP-N1-cortactin (Cort), pEGFP-N1-Cort-Y421A, pEGFP-N1-Cort-Y470A and pEGFP-N1-Cort-Y486A. The cells were divided into a normal control group, a shear stress group, a shear stress + pEGFP-N1 group, a shear stress + PEGFP-N1-Cort group, a shear stress + pEGFP-N1-Cort-Y421A group, a shear stress + pEGFP-N1-Cort-Y470A group, and a shear stress + pEGFP-N1-Cort-Y486A group. The shear stress were set at 4 dynes/cm2. The levels of MUC5AC protein and mRNA in cells and culture supernatant were assayed with enzyme-linked immunosorbent assay (ELISA) and real-time PCR. The cortactin and phosphorylated cortactin were detected by Western blot. F-actin was stained by fluorescein isothiocyanate (FITC)-phalloidin.
Results: There was an obvious increase of phosphorylated cortactin in cells exposed to 4 dynes/cm2 of shear stress for 30 min, which reached climax at 2 hours concomitant with elevation of MUC5AC protein production and mRNA expression in the different experiment groups (all P<0.05). Compared with single shear stress-stimulated group, MUC5AC in supernatant was increased obviously, and the distribution of F-actin in cytomembrane was also increased in the pEGFP-N1-Cort group (both P<0.05), while there were no changes in the MUC5AC protein and mRNA levels in cytoplasm. Compared with the shear stress+pEGFP-N1-Cort group, the MUC5AC protein in the culture supernatant was decreased, and the polymerization of F-actin at cell membranes were also attenuated in the shear stress+pEGFP-N1-Cort-Y421A group and the shear stress + pEGFP-N1-Cort-Y470A group (both P<0.05), while there was no significant effect in the shear stress + pEGFP-N1-Cort-Y486A group (P>0.05).
Conclusion: Cortactin is involved in shear stress-mediated MUC5AC secretion in human airway epithelial cells, and the phosphorylated site of Tyr421 and Tyr470 may play an important role in it.
Cortactin
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Epithelial Cells
;
Humans
;
Mucin 5AC
;
Mucus
;
Phosphorylation
2.Effects of Curcumin and Genistein on Phorbol Ester or Tumor Necrosis Factor-alpha-Induced Mucin Production from Human Airway Epithelial Cells.
Su Yel LEE ; Hyun Jae LEE ; Jae Woo LEE ; Byeong Kyou JEON ; Ju Ock KIM ; Choong Jae LEE
Tuberculosis and Respiratory Diseases 2011;70(3):218-223
BACKGROUND: We investigated whether curcumin and genistein affect the MUC5AC mucin production from human airway epithelial cells that is induced by phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-alpha (TNF-alpha). METHODS: Confluent NCI-H292 cells were pretreated with each agent for 30 min and then stimulated with PMA or TNF-alpha for 24 hours. MUC5AC mucin production was measured by an ELISA. RESULTS: (1) Curcumin dose-dependently inhibited the production of MUC5AC mucin that was induced by PMA or TNF-alpha; (2) Genistein inhibited PMA-induced MUC5AC mucin production. However, it did not decrease TNF-alpha-induced MUC5AC mucin production. CONCLUSION: These results suggest that curcumin and genistein inhibit the production of airway mucin induced by PMA.
Curcumin
;
Epithelial Cells
;
Genistein
;
Humans
;
Mucin 5AC
;
Mucins
;
Necrosis
;
Phorbols
;
Tumor Necrosis Factor-alpha
3.Menthol enhances interleukin-13-induced synthesis and secretion of mucin 5AC in human bronchial epithelial cells.
Mingyang ZHANG ; Jing WANG ; Minchao LI
Journal of Southern Medical University 2020;40(10):1432-1438
OBJECTIVE:
To investigate the effect of interleukin (IL) -13 combined with cold stimulation on synthesis and secretion of mucin (MUC) 5AC in human bronchial epithelial cell line 16HBE and explore the role of transient receptor potential 8 (TRPM8) and anti-apoptotic factor B-cell lymphoblast-2 (Bcl-2) in this process.
METHODS:
16HBE cells were stimulated with 10 ng/mL IL-13, 1 mmol/L menthol, or both (1 mmol/L menthol was added after 6 days of IL-13 stimulation), and the changes in the expression of MUC5AC, intracellular Ca
RESULTS:
The mRNA and protein expressions of MUC5AC increased significantly in 16HBE cells following stimulation with IL-13, menthol, and both (
CONCLUSIONS
Menthol combined with IL-13 produces a synergistic effect to promote the synthesis and secretion of MUC5AC in 16HBE cells possibly by activating TRPM8 receptor to upregulate the expression of Bcl-2.
Bronchi
;
Epithelial Cells/drug effects*
;
Humans
;
Interleukin-13
;
Menthol/pharmacology*
;
Mucin 5AC
4.Dickkopf-1 inhibits the secretion of MUC5AC induced by Mycoplasma pneumoniae P1-C in mouse lung epithelial cells.
Juan SHI ; Chunji MA ; Xiujing HAO ; Haixia LUO ; Min LI
Chinese Journal of Biotechnology 2023;39(1):248-261
Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.
Animals
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Mice
;
Epithelial Cells
;
Lung
;
Mucin 5AC/metabolism*
;
Mycoplasma pneumoniae/metabolism*
;
Signal Transduction
5.Expression of MUC3, MUC5AC, MUC6 and Epidermal Growth Factor Receptor in Gallbladder Epithelium according to Gallstone Composition.
Hyo Jung KIM ; Jae Seon KIM ; Kyoung Oh KIM ; Ki Ho PARK ; Hyung Joon YIM ; Jin Yong KIM ; Jong Eun YEON ; Jong Jae PARK ; Jae Jeong SHIM ; Kwan Soo BYUN ; Young Tae BAK ; Chang Hong LEE
The Korean Journal of Gastroenterology 2003;42(4):330-336
BACKGROUND/AIMS: Gallbladder (GB) mucin is one of the key factors in the gallstone formation. However, there is little information about the diversity of mucin secretion according to the stone composition. Epidermal growth factor receptor (EGFR) functions in proliferation including mucin secreting goblet cell hyperplasia. We compared the expressions of MUC3, MUC5AC, MUC6 and EGFR in the GB epithelium with cholesterol gallstones (GB-chol) group and pigment gallstones (GB-pig group). METHODS: GBs from elective laparoscopic cholecystectomy for the gallstone disease were studied. Stone composition was analyzed by the spectrophotometer. Immunohistochemical stain was performed using each monoclonal antibody. The percentage of stained proportion was scored by the NIH image program and the results were compared between both groups. RESULTS: Total 20 patients were enrolled (10 patients with cholesterol gallstones, 10 patients with pigment gallstones). The percentages of stained proportion for MUC3, MUC5AC, and MUC6 were 42+/-27%, 31+/-15%, and 17+/-9%, respectively in GB-chol group and 32+/-22%, 33+/-23%, and 15+/-10%, respectively in GB-pig group (p>0.05). The expression of EGFR was 50% (5/10) in the GB-chol group and 80% (8/10) in the GB-pig group respectively. CONCLUSIONS: There was no difference in the expressions of MUC3, MUC5AC, and MUC6 between the two groups. Further studies are needed to elucidate the role of EGFR in the gallstore formation.
Bile Pigments/analysis
;
Cholelithiasis/chemistry/*metabolism
;
Cholesterol/analysis
;
Epithelium/metabolism
;
Gallbladder/*metabolism
;
Humans
;
Immunohistochemistry
;
Mucin 5AC
;
Mucin-3
;
Mucin-6
;
Mucins/*analysis
;
Receptor, Epidermal Growth Factor/*analysis
6.Regulation of Tumor Necrosis Factor-alpha-induced Airway Mucin Production and Gene Expression by Carbenoxolone, Prunetin, and Silibinin.
Hyun Jae LEE ; Su Yel LEE ; Byeong Kyou JEON ; Jae Woo LEE ; Mi Nam LEE ; Ju Ock KIM ; Choong Jae LEE
Tuberculosis and Respiratory Diseases 2010;69(5):348-353
BACKGROUND: In this study, we tried to investigate whether carbenoxolone, prunetin, and silibinin affect tumor necrosis factor (TNF)-alpha-induced MUC5AC mucin production and gene expression from human airway epithelial cells. METHODS: Confluent NCI-H292 cells were pretreated with each agent (carbenoxolone, prunetin, and silibinin) for 30 min and then stimulated with TNF-alpha for 24 hours. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively. RESULTS: Carbenoxolone, prunetin and silibinin inhibited the production of MUC5AC mucin protein induced by TNF-alpha; the 3 compounds also inhibited the expression of MUC5AC mucin gene induced by TNF-alpha. CONCLUSION: This result suggests that carbenoxolone, prunetin and silibinin can inhibit mucin gene expression and production of mucin protein induced by TNF-alpha, by directly acting on airway epithelial cells.
Carbenoxolone
;
Enzyme-Linked Immunosorbent Assay
;
Epithelial Cells
;
Gene Expression
;
Humans
;
Isoflavones
;
Mucin 5AC
;
Mucins
;
Necrosis
;
Silymarin
;
Tumor Necrosis Factor-alpha
8.Effects of respiratory syncytial virus infection on epidermal growth factor receptor, tight junction association proteins and mucin in airway epithelial cells.
Juan-Juan LIU ; Ting ZHANG ; Yu-Mei MI
Chinese Journal of Contemporary Pediatrics 2019;21(3):294-299
OBJECTIVE:
To study the effects of respiratory syncytial virus (RSV) infection on epidermal growth factor receptor (EGFR), tight junction association proteins and mucin in the human airway epithelial cells.
METHODS:
Human airway epithelial cells NCI-H292 were randomly treated by ultraviolet light-inactivated RSV (control group) or thawed RSV (RSV infection group). After 48 hours of treatment, the protein levels of occludin, E-cadherin, phosphorylated EGFR and EGFR in NCI-H292 cells were measured by Western blot. The distribution and expression levels of occludin and E-cadherin in NCI-H292 cells were examined by immunofluorescence technique. The expression levels of MUC5AC mRNA in NCI-H292 cells were assessed by RT-PCR.
RESULTS:
The protein levels of occludin and E-cadherin were significantly reduced in the RSV infection group compared with the control group (P<0.05). The protein levels of phosphorylated EGFR and EGFR increased significantly in the RSV infection group compared with the control group (P<0.05). The MUC5AC mRNA levels also increased significantly in the RSV infection group compared with the control group (P<0.05).
CONCLUSIONS
RSV may down-regulate the tight junction association proteins and up-regulate the expression of MUC5AC in airway epithelial cells, which contributes to epithelial barrier dysfunction. EGFR phosphorylation may play an important role in regulation of airway barrier.
Cell Line
;
Epithelial Cells
;
ErbB Receptors
;
Humans
;
Mucin 5AC
;
Respiratory Syncytial Virus Infections
;
Tight Junction Proteins
;
Tight Junctions
9.The expression of mucins gene in the human nasal polyps and allergic rhinitis.
Journal of Clinical Otorhinolaryngology Head and Neck Surgery 2009;23(20):923-929
OBJECTIVE:
To detect the mucin gene (MUC2, MUC5AC, MUC5B, MUC18 and MUC19) expression in the nasal polyps, allergic rhinitis (AR) and the normal nasal mucosa in human. To investigate the role and clinical significance of mucin gene in the pathogenesis of nasal polyps and AR patients.
METHOD:
We obtained samples from 35 cases of nasal polyps, 18 cases of AR inferior turbinate and 18 cases of simple nasal septum deviation inferior turbinate. Specimens were analyzed with RT-PCR and Real-time FQ-RT-PCR.
RESULT:
The results of RT-PCR and FQ-RT-PCR showed that the expression of MUC5AC, MUC5B in nasal polyps and AR patients was significantly higher than that in normal mucosa (P<0.05). The expression of MUC5AC, MUC5B in nasal polyps was not significantly different from that in AR patients (P>0.05). The expression of MUC2, MUC18 in nasal polyps and AR was not significantly different from that in normal mucosa (P>0.05). And the results of RT-PCR for MUC19 expression in AR was higher than that in nasal polyps group and normal group (P<0.05 or P<0.01).
CONCLUSION
MUC5AC and MUC5B are highly expressed in epithelium of human nasal polyps and AR, and they take part in mucus over-secretion in nasal polyps and AR. The expression of MUC19 in AR was higher than that in nasal polyps group and normal group. It indicates that the secretion of MUC19 in allergic rhinitis was on high level. There was no difference of the expression of MUC2 and MUC18 in nasal polyps group, AR group and in normal group.
Adolescent
;
Adult
;
Female
;
Gene Expression
;
Humans
;
Male
;
Middle Aged
;
Mucin 5AC
;
genetics
;
Mucin-2
;
genetics
;
Mucin-5B
;
genetics
;
Mucins
;
genetics
;
metabolism
;
Nasal Mucosa
;
metabolism
;
pathology
;
Nasal Polyps
;
genetics
;
metabolism
;
Rhinitis
;
genetics
;
metabolism
;
Young Adult
10.Cholesterol Depletion in Cell Membranes of Human Airway Epithelial Cells Suppresses MUC5AC Gene Expression.
Kee Jae SONG ; Na Hyun KIM ; Gi Bong LEE ; Ji Hoon KIM ; Jin Ho KWON ; Kyung Su KIM
Yonsei Medical Journal 2013;54(3):679-685
PURPOSE: If cholesterol in the cell membrane is depleted by treating cells with methyl-beta-cyclodextrin (MbetaCD), the activities of transmembrane receptors are altered in a cell-specific and/or receptor-specific manner. The proinflammatory cytokines, IL-1beta is potent inducers of MUC5AC mRNA and protein synthesis in human airway epithelial cells. Cells activated by IL-1beta showed increased phosphorylation of extracellular signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Thus, we investigated the effects of cholesterol depletion on the expression of MUC5AC in human airway epithelial cells and whether these alterations to MUC5AC expression were related to MAPK activity. MATERIALS AND METHODS: After NCI-H292 cells were pretreated with 1% MbetaCD before adding IL-1beta for 24 hours, MUC5AC mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and real time-PCR. Cholesterol depletion by MbetaCD was measured by modified microenzymatic fluorescence assay and filipin staining. The phosphorylation of IL-1 receptor, ERK and p38 MAPK, was analyzed by western blot. RESULTS: Cholesterol in the cell membrane was significantly depleted by treatment with MbetaCD on cells. IL-1beta-induced MUC5AC mRNA expression was decreased by MbetaCD and this decrease occurred IL-1-receptor-specifically. Moreover, we have shown that MbetaCD suppressed the activation of ERK1/2 and p38 MAPK in cells activated with IL-1beta. This result suggests that MbetaCD-mediated suppression of IL-1beta-induced MUC5AC mRNA operated via the ERK- and p38 MAPK-dependent pathway. CONCLUSION: Cholesterol depletion in NCI-H292 cell membrane may be considered an anti-hypersecretory method since it effectively inhibits mucus secretion of respiratory epithelial cells.
Cell Membrane/drug effects/*metabolism
;
Cholesterol/*metabolism
;
Epithelial Cells/metabolism
;
Gene Expression
;
Humans
;
Mucin 5AC/genetics/*metabolism
;
Respiratory System/*metabolism/pathology
;
beta-Cyclodextrins/pharmacology