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

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

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.
Animals ; Ear, Middle/*metabolism/pathology ; Mice ; Microscopy, Electron, Scanning ; Mucin 5AC/genetics/*metabolism ; RNA, Messenger/*genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Spectrophotometry

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.
Animals ; Ear, Middle/*metabolism/pathology ; Mice ; Microscopy, Electron, Scanning ; Mucin 5AC/genetics/*metabolism ; RNA, Messenger/*genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Spectrophotometry

OBJECTIVE: To investigate the mechanism of activator protein-1 (AP-1) on cigarette smoke-induced airway mucous hypersecretion and to explore the possible signal transduction pathway that activates AP-1. METHODS: The airway epithelial cell line (BEAS-2B) was cultured in vivo and treated with cigarette smoke extract (CSE). The DNA binding activity of AP-1 was blocked by the transfection of c-Jun dominant negative mutant TAM67 into the cells. SP600125 and PD98059 were used to block the activation of c-Jun terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) respectively. MUC5AC protein was detected by enzyme-linked immunosorbent assay, MUC5AC mRNA level was analyzed by RT-PCR, while the protein contents of p-JNK, p-ERK and p-P38 were detected by Western blot, and the DNA binding activity of AP-1 was determined by electrophoretic mobility shift assay. RESULTS: The MUC5AC protein production and mRNA expression in the CSE group were significantly higher than those in the control group, and the DNA binding activity of AP-1 was also higher than that in the control group (P<0.01). The protein contents of p-ERK and p-JNK in the CSE group were higher than those in the control group (P<0.01), but the p-P38 level was not significantly different from that in the control group (P>0.05). After the transfection of TAM67 into the cells, the expression levels of MUC5AC protein and mRNA and the binding activity of AP-1 decreased significantly (P<0.01). The DNA binding activity of AP-1 and the expression levels of MUC5AC protein and mRNA were lower in the SP600125 group and in the PD98059 group than those in the CSE group (P<0.05). CONCLUSION After being activated by JNK and ERK which are phosphorylated by cigarette smoke, AP-1 binds to its DNA binding elements on the promoter of MUC5AC gene and up-regulates the MUC5AC expression at the transcriptional level.
Bronchi ; cytology ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Humans ; Mucin 5AC ; genetics ; metabolism ; Smoke ; adverse effects ; Smoking ; adverse effects ; Tobacco ; chemistry ; Transcription Factor AP-1 ; pharmacology

BACKGROUNDExpression of murine calcium-activated chloride channel family member 3 (mCLCA3) has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin (OVA). However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified.

METHODSmCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid (BALF) were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC (MUC5AC) were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 (IL-13) were determined quantitatively.

RESULTSmCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-γ, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group (P < 0.05). The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALF. The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue.

CONCLUSIONThese findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may involve mCLCA3 overexpression up-regulating IL-13 expression.

Allergens ; Animals ; Asthma ; Chloride Channels ; Female ; Inflammation ; chemically induced ; metabolism ; Interleukin-13 ; metabolism ; Interleukin-4 ; genetics ; metabolism ; Interleukin-5 ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Mucin 5AC ; genetics ; metabolism ; Ovalbumin ; pharmacology

OBJECTIVETo investigate the effect of interleukin-13 (IL-13) on mucus secretion in vivo and the possible mechanism.

METHODSThe SD rats were randomly divided into control group, IL-13 group and IL-13 plus SP600125 group. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and the level of MUC5AC in the lung tissues were examined using Western blotting. RT-PCR was performed to examine the mRNA level of STAT4 and STAT6, and electrophoretic mobility shift assays (EMSA) was used to detect the DNA-binding activities of Forkhead box a2 (FOXA2) and activator protein-1 (AP-1).

RESULTSIL-13 caused a significant increase in MUC5AC and p-JNK1/2 expression, but did not affect the phosphorylation of ERK1/2. The expression of MUC5AC was attenuated after treatment with SP600125. A significant increase in STAT6 was observed in IL-13 group compared with that in the control group, whereas the expression of STAT4 mRNA was not significantly affected. The DNA-binding activity of FOXA2 was down-regulated after IL-13 exposure, which did not affect the DNA-binding activity of AP-1.

CONCLUSIONIL-13 down-regulates mucus secretion via STAT6-FOXA2 pathway in vitro.

Animals ; Bronchi ; secretion ; Female ; Hepatocyte Nuclear Factor 3-beta ; genetics ; metabolism ; Interleukin-13 ; pharmacology ; Male ; Mucin 5AC ; metabolism ; Mucus ; secretion ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; STAT6 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; drug effects

Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; genetics ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Mucin 5AC ; genetics ; secretion ; RNA, Messenger ; genetics ; secretion ; Respiratory System ; cytology ; Tumor Necrosis Factor-alpha ; pharmacology ; Up-Regulation ; drug effects

BACKGROUNDEndoplasmic reticulum (ER) stress and ER stress-mediated apoptosis were reported to be involved in the pathogenesis of several diseases. In a recent study, it was reported that the ER stress pathway was activated in the lungs of lipopolysaccharide (LPS)-treated mice. It was also found that the C/EBP homologous protein (CHOP), an apoptosis-related molecule, played a key role in LPS-induced lung damage. The aim of this study was to verify whether LPS could activate the ER stress response in airway epithelial cells and which molecule was involved in the pathway. This study was also aimed at finding new reagents to protect the airway epithelial cells during LPS injury.

METHODSER stress markers were observed in LPS-incubated NCI-H292 cells. SiRNA-MUC5AC was transfected into NCI-H292 cells. The effects of dexamethasone and erythromycin were observed in LPS-induced NCI-H292 cells.

RESULTSLPS incubation increased the expression of ER stress markers at the protein and mRNA levels. The knockout of MUC5AC in cells attenuated the increase in ER stress markers after incubation with LPS. Dexamethasone and erythromycin decreased caspase-3 activity in LPS-induced NCI-H292 cells.

CONCLUSIONSLPS may activate ER stress through the overexpression of MUC5AC. Dexamethasone may protect human airway epithelial cells against ER stress-related apoptosis by attenuating the overload of MUC5AC.

Animals ; Apoptosis ; drug effects ; genetics ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Dexamethasone ; pharmacology ; Endoplasmic Reticulum ; drug effects ; metabolism ; Erythromycin ; pharmacology ; Humans ; Lipopolysaccharides ; pharmacology ; Mice ; Mucin 5AC ; genetics ; metabolism ; RNA, Small Interfering

OBJECTIVE: To study the effects of dexamethasone and histamine on the expression of MUC5AC mRNA and protein in human nasal polyps. METHOD: All samples were randomly divided into control group, histamine stimulating group and dexamethasone group. The expression of MUC5AC mRNA and MUC5AC protein in nasal polyps were respectively detected by the methods of reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical assay. RESULT: The expression of MUC5AC mRNA (0.6389 +/- 0.0526) and protein (0.1934 +/- 0.0137) in histamine stimulating group were significantly higher than those of control group (0.3495 +/- 0.0357 and 0.1172 +/- 0.0173, respectively) and reduced (0.4988 +/- 0.0603 and 0.1444 +/- 0.0075, respectively) after treatment with dexamethasone. CONCLUSION Histamine upregulates MUC5AC mRNA and protein expression. Dexamethasone downregulates MUC5AC mRNA and protein expression, which may be one of the mechanisms of downregulating mucus overproduction by dexamethasone in nasal polyps.
Adolescent ; Adult ; Dexamethasone ; pharmacology ; Female ; Glucocorticoids ; pharmacology ; Histamine ; pharmacology ; Humans ; Male ; Middle Aged ; Mucin 5AC ; genetics ; metabolism ; Nasal Mucosa ; metabolism ; Nasal Polyps ; metabolism ; RNA, Messenger ; genetics ; Up-Regulation ; Young Adult