MUC5AC Protein Research Articles

Mediated mechanism of NDRG2 in the regulation of neutrophil nuclear transcription factor-κB signaling pathway through the deubiquitylating enzyme of Zc3h12d

To explore the role NDRG2/Zc3h12d in the regulation of neutrophil nuclear transcription factor-κB signaling pathway and the underlying mechanisms. Sixteen HBE cells were incubated with lipopolysaccharide (LPS) to establish airway mucus hypersecretion model, which was transfected with NDRG2 or Zc3h12d siRNA. The cells were divided into 5 groups: a LPS+NDRG2 siRNA (Group A), a LPS+ NDRG2 and Zc3h12d siRNAs (Group B), a LPS+Zc3h12d siRNA (Group C), a LPS+ empty plasmid (Group D), and a negative control group (Group E). The reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression level of mucin (MUC) 5AC mRNA. The levels of MUC5AC and the inflammatory factors were examined by the enzyme-linked immunosorbent assay (ELISA). The NF-κB p65 and the Zc3h12d protein levels were measured by Western blot. The MUC5C expression was further examined by laser confocal method. Compared with Group B, the levels of MUC5AC mRNA and protein in Group A were decreased (P<0.05), there was no significant difference in the MUC5AC mRNA and MUC5AC protein levels between the Group B and the Group C (P>0.05). Compared with Group D, the MUC5AC (mRNA and protein) and inflammatory factor levels in the Group A were significantly decreased (P<0.05); compared with Group E, after incubation with LPS, the levels of MUC5AC (mRNA and protein) and inflammatory factors in the Group D was increased, with significantly difference (P<0.05). NDRG2 can regulate the function of NF-κB signaling pathway through the deubiquitylating enzyme of Zc3h12d.

SUPPRESSIVE EFFECTS OF WELSH ONION EXTRACTS ON MUCUS HYPER-PRODUCTION IN HUMAN AIRWAY CELLS

We investigated the suppressive effects of Welsh onion (Allium fistulosum L.) on the hyper-production of mucins in NCI-H292 human lung cancer cells. Periodic acid-Schiff staining showed that the 50% (v/v) ethanol extract of Welsh onion suppressed mucus glycoprotein production in cells, whereas the water and ethanol extracts did not. A real-time RTPCR analysis demonstrated that the 50% ethanol extract attenuated the gene expression of MUC2 and MUC5AC, which are major airway mucus components, in NCI-H292 cells stimulated with lipopolysaccharide (LPS). Furthermore, dot blot hybridization revealed that the extract at 100 μg/ml attenuated the hyper-production of the MUC5AC protein in LPS-stimulated NCI-H292 cells.

Temporal and spatial expression of Muc2 and Muc5ac mucins during rat respiratory and digestive tracts development

Secreted mucins constitute a crucial part of the gel that protects respiratory and digestive epithelia, being MUC2/Muc2 the predominant gel-forming mucin of the intestine while MUC5AC/Muc5ac is one of the gel-forming mucins most expressed at the airways. In this study, we have analyzed Muc2 and Muc5ac during rat development by using immunohistochemistry, Western blotting and RT-PCR. We demonstrated that rat Muc2 was expressed in fetal intestinal goblet cells of surface epithelium of villi and developing Lieberkühn crypts. In neonates and adults, Muc2 was expressed at luminal goblet cells of small and large intestine and at gastric mucous and glandular cells. Muc5ac protein was observed in embryonic gastric and lung samples; expression increased during development and postnatal and adult life. After birth, a low reaction was detected at the tracheal surface epithelium and glands, which increased in adults.

HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells

Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, and Western blotting was performed to evaluate signaling transduction pathway. Hemin (4μM) significantly increased HO-1 protein expression (p<0.01) and HO-1 mRNA expression (p<0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p<0.01) and MUC5AC mRNA (p<0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p<0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p<0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression.In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.

Verproside inhibits TNF-α-induced MUC5AC expression through suppression of the TNF-α/NF-κB pathway in human airway epithelial cells

Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of MUC5AC, are significant risk factors in asthma and chronic obstructive pulmonary disease (COPD) patients. Previously, we reported that verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a potent anti-asthmatic candidate drug in vivo. However, the molecular mechanisms underlying the pharmacological actions of verproside remain unknown.Here, we found that verproside significantly reduces the expression levels of tumor necrosis factor alpha (TNF-α)-induced MUC5AC mRNA and protein by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors such as IκB kinase (IKK)β, IκBα, and TGF-β-activated kinase 1 (TAK1) in NCI-H292 cells. Moreover, verproside attenuated TNF-α-induced MUC5AC transcription more effectively when combined with an IKK (BAY11-7082) or a TAK1 (5z-7-oxozeaenol) inhibitor than when administered alone. Importantly, we demonstrated that verproside negatively modulates the formation of the TNF-α-receptor (TNFR) 1 signaling complex [TNF-RSC; TNFR1-recruited TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), receptor-interacting protein kinase 1 (RIP1), and TAK1], the most upstream signaling factor of NF-κB signaling. In silico molecular docking studies show that verproside binds between TRADD and TRAF2 subunits.Altogether, these results suggest that verproside could be a good therapeutic candidate for treatment of inflammatory airway diseases such as asthma and COPD by blocking the TNF-α/NF-κB signaling pathway.

15-Hydroxyeicosatetraenoic Acid Inhibits Phorbol-12-Myristate-13-Acetate-Induced MUC5AC Expression in NCI-H292 Respiratory Epithelial Cells

It has been reported that overexpression of MUC5AC induced by excessive inflammation leads to airway obstruction in respiratory diseases such as chronic obstructive pulmonary disease and asthma. 15-Hydroxyeicosatetraenoic acid (15-HETE) has been reported to have anti-inflammatory effects, but the role of 15-HETE in respiratory inflammation has not been determined. Therefore, the aim of this study was to investigate the effects of 15-HETE on MUC5AC expression and related pathways. In this study, phorbol-12-myristate-13-acetate (PMA) was used to stimulate NCI-H292 bronchial epithelial cells in order to examine the effects of 15-HETE. 15-HETE inhibited PMA-induced expression of MUC5AC mRNA and secretion of MUC5AC protein. Also, 15-HETE regulated matrix metallopeptidase 9 (MMP-9), mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK). In addition, 15-HETE decreased translocation of specificity protein-1 (Sp-1) transcription factor and nuclear factor kappaB (NF-κB) into nuclear. Furthermore, 15-HETE enhanced transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) as a PPARγ agonist. This activity reduced phosphorylation of protein kinase B (PΚB/Akt) by increasing the expression of phosphatase and tensin homolog (PTEN). In conclusion, 15-HETE regulated MUC5AC expression via modulating MMP-9, MEK/ERK/Sp-1 and PPARγ/PTEN/ Akt signaling pathways in PMA-treated respiratory epithelial cells.

Induction of MUC5AC mucin expression by histamine through the activation of its core promoter region

Conclusion: This study provides evidence that histamine induced MUC5AC mRNA expression through the activation of the core region of its promoter. It may also help in approaching new therapeutic strategies in airway mucins hypersecretory diseases. Objective: Mucin hypersecretion characterizes several respiratory diseases. Production of MUC5AC, a major gel forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators. Histamine is associated with MUC5AC up-regulation during the early phase of allergic respiratory diseases. The goal of the present study was to identify whether histamine may induce MUC5AC gene expression both at mRNA and protein levels and to elucidate its mechanism. Methods: Guinea pigs were sensitized and challenged with dermatophagoides farinae (Der f) extract. Human lung mucoepidermoid carcinoma cell line (NCI-H292) was used. The regulatory mechanism of MUC5AC by histamine and H1R was investigated using RT-PCR, immunofluorescence, and MUC5AC promoter-driven luciferase reporter assay. Results: The MUC5AC expression levels were increased by histamine treatment in either nasal tissues of Der f challenged guinea pigs or NCI-H292 cells, whereas the MUC5AC protein over-production induced by histamine administration was significantly inhibited by H1R antagonist chlorpheniramine. It was found that histamine enhanced the activation of the proximal core region of the MUC5AC promoter, which was significantly blocked by chlorpheniramine, as indicated by luciferase reporter assays.

Mo1973 Immunohistochemical Markers Differentiating Colonic Sessile Serrated Adenomas/Polyps (SSA/Ps) From Hyperplastic Polyps

Differentiating SSA/Ps from hyperplastic polyps (HPs), with little or no risk for progression to cancer, can be a major challenge using conventional endoscopic and histological examinations. SSA/Ps are frequently right sided, flat and have a mucus cap. The goal of this study was identifying potential immunohistochemical (IHC) markers that help to differentiate SSA/ Ps from HPs. Methods: We compared RNA sequencing transcriptional signatures of SSA/Ps (n=15) and control (n=20) colonic mucosa from patients undergoing screening colonoscopy. Reads from the analysis were aligned to the GRCh37/Hg19 human reference genome and filtered for high quality alignments (http://novocraft.com). Hierarchical clustering of log2 ratios comparing SSA/Ps and controls were performed and mined for candidate IHC targets. IHC analysis was performed on formalin-fixed paraffin-embedded sections of 12 SSA/Ps, 10 HPs, 5 adenomas, 5 colon cancers and 5 control colon specimens to assess staining for selected antigens. Expression of MUC5AC and TFF1 protein were quantified with ImageJ software. Results: Bioinformatics analysis identified 1,294 (≥1.5-fold change) differentially expressed annotated genes (false discovery rate (FDR) <0.05) in SSA/Ps as compared to normal controls. The dataset was mined for the most highly expressed genes and genes with possible biological functions related to SSA/Ps. A total of 249 genes were overexpressed ≥5fold in SSA/Ps compared to controls. SSA/Ps markedly overexpressed MUC5AC (582-fold), a component of the gastric mucus layer, and a secretory protein that may stabilize mucin layers, trefoil factor 1 (TFF1, 79-fold). Neither protein is typically expressed in normal colonic mucosa. SSA/Ps exhibited strong co-localized staining for both proteins (Fig. 1 and 2). Intense MUC5AC staining was primarily seen in the serrated crypts, whereas expression in adjacent non-serrated mucosa was identical to controls. Conversely, HPs showed only weak patchy staining of MUC5AC and variable TFF1 staining that was infrequently colocalized. Normal colon had no detectable MUC5AC staining and minimal TFF1 staining. Conventional adenomas showed trace MUC5AC and TFF1 staining. Conclusions: RNA-seq and IHC analysis showed markedly increased expression of two mucin-associated genes/ proteins in SSA/Ps, MUC5AC and TFF1, which are not typically expressed in normal colonic mucosa or adenomas. There was also intense co-localization of MUC5AC and TFF1 in SSA/ Ps but not in HPs. These results identify intense co-localized expression of MUC5AC and TFF1 as a possible diagnostic marker that would aid differentiating SSA/Ps from HPs. Further studies will be needed with larger numbers of SSA/Ps. Since MUC5AC has been linked to signaling related to bacteria, these findings suggest that luminal factors may play a role in the formation or progression of SSA/Ps.

Role and mechanism of heat shock protein 70 in airway hypersecretion

To explore the role and mechanism of heat shock protein 70 (HSP70) in airway hypersecretion. After a stimulation of 8% cigarette smoke extract (CSE), airway cells A549 were treated with HSP70 antibody and c-Jun N-terminal kinase (JNK) specific inhibitor SP600125 respectively. And the cells were divided into 4 groups of blank contrast (serum-free medium), CSE stimulation (8% CSE for 24 h), HSP70 antibody (30-min pre-treatment of HSP70 antibody and culturing in 8% CSE for 24 h) and SP600125 (30-min pre-treatment of SP600125 30 µmol/L and culturing in 8% CSE for 24 h). The relative expression levels of MUC5AC protein in various groups were determined by enzyme-linked immunosorbent assay (ELISA). And the relative transcription level of MUC5AC mRNA was detected by reverse transcription-polymerase chain reaction (PCR) while the synthesized levels of HS70 as well as the phosphorylation levels of JNK and activated protein-1 (mostly c-Jun) were measured by Western blot. As compared with those in blank contrast group (0.26 ± 0.10, 0.28 ± 0.06, 0.30 ± 0.05, 0.30 ± 0.08, 0.36 ± 0.08), HSP70 antibody group (0.30 ± 0.12, 0.29 ± 0.09, 0.34 ± 0.06, 0.47 ± 0.19, 0.39 ± 0.13) and SP600125 group (0.38 ± 0.06, 0.31 ± 0.14, 0.39 ± 0.04, 0.44 ± 0.12, 0.48 ± 0.11), the relative expression levels of MUC5AC protein and mRNA, phosphorylation JNK (p-JNK), c-Jun and p-c-Jun (0.52 ± 0.07, 0.64 ± 0.11, 0.73 ± 0.06, 0.67 ± 0.10, 0.67 ± 0.09) significantly increased in CSE stimulation group (all P < 0.05). And the synthesis levels of HSP70 (0.75 ± 0.09) in CSE stimulation group increased than blank contrast group (0.29 ± 0.03) and HSP70 antibody group (0.40 ± 0.11) (all P < 0.05). HSP70 may enhance the expression of MUC5AC in bronchial epithelial A549 cells via a signaling pathway of JNK/AP-1..

Naringenin may block RSV-induced mucous hypersecretion in A549 cell via JNK/AP-1 signaling pathway

Naringenin has been reported to attenuate Mucin (MUC) 5AC secretion in many pathological models. Many stimuli activate MUC5AC expression through JNK/AP-1 signaling pathways. We hypothesized that naringenin may have inhibitory effects on mucous hypersecretion by modulating MUC5AC production and inhibiting JNK/AP-1 signaling pathways. The cell model of mucous hypersecretion was made by human lung adenocarcinoma epithelial (A549) cells stimulated by RSV. A549 cells were subcultured and then randomly divided into 7 groups, which were designated as group C (cell control group), groups R1-3 (cells were infected with RSV at the multiplication of infection (MOI) of 0. 5, 1. 0, 5. 0), groups N1-2 (cells infected with viruses in presence of Nar 30 - 100 mol/L), groups N3-4 (uninfected cells treated with Nar 30 - 100 µmol/L), group D (DMSO), group S (cells infected with viruses in presence of SP600125). After incubating for 24 hrs, the expression of MUC5AC at mRNA and protein level in the groups were determined by real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA). The protein expression changes of JNK, p-JNK and AP-1 were measured by Western blotting. The expressions of MUC5AC protein and mRNA in all RSV infected groups were significantly higher than that in group C in a dose-dependent manner (all P <0. 05). Nar of 30 and 100 µmol/L significantly and dose-dependently decreased RSV-induced secretion of MUC5AC protein in cell supernatant and expression of MUC5AC mRNA (P <0. 05). The relative content of p-JNK, AP-l in R2 groups were 3. 31 ± 0. 34 and 1. 94 ± 0. 05. Theyfrweremtgnificanty increased as compared with group C (both 1. 00 ± 0. 00) (all P <0. 05). The levels of p-JNK in N2 and S groups were 2. 10 ± 0. 20. 27 and 1.±97 ± 0. 16. The levels of AP-1 in N2 and S groups were 1. 40 ± 0. 03, 1. 36 ± 0. 05. Nar and SP600125 led to a largest decrease in levels of p-JNK and AP-1 when compared with group R2 (P <0. 05). The MUC5AC protein in group R2 was (48. 19 ± 0. 47) µg/L. The protein expression of MUC5AC in group R2 was significantly higher than that in group C [(36. 67 ± 1. 50) g/L] with a statistically significant difference (P <0. 05). The protein expression of MUC5AC in groups N2 and S were(43. 17 ± 1. 06) µg/L, (44.±02 ± 0. 99) µg/L, Nar and SP600125 remarkably inhibited RSV-induced secretion of MUC5AC in supernatant of A549 cells (P < 0. 05). Naringenin might be able to block RSV-induced mucous

Muc5ac mucin expression during rat skin development.

Some mucin genes have been detected during human embryonic and fetal organ development; however, little is known about mucin expression in epidermal development, neither in humans nor in other species. The present research was developed to explore Muc5ac skin expression during pre- and post-natal rat development. Immunohistochemistry (IHC), Western blotting (WB) and RT-PCR were employed. By IHC, Muc5ac protein was found early in embryonic epidermis from day 13 of gestation until seven days after birth when the surface epidermis became negative and the reaction was restricted to secreting sebum cells. In coincidence with IHC findings, WB analysis showed a band at approximately 200KDa at the same periods of development. Results were also confirmed by RT-PCR.Muc5ac expression in rat embryonic epidermis suggests that Muc5ac may play a protective role in embryonic skin previous to birth which may be replaced by pile covering. To our knowledge, this is the first report that confirmed Muc5ac expression during skin development.

Impact of Staphylococcus Epidermidis Lysates on Middle Ear Epithelial Proinflammatory and Mucogenic Response

BackgroundChronic otitis media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Staphylococcus epidermidis, typically considered a commensal...

Leukotriene E4 induces MUC5AC release from human airway epithelial NCI-H292 cells

BackgroundHypersecretion of mucin in the airway epithelium is an important feature of allergic airway diseases. Of the 3 cysteinyl leukotrienes (CysLTs; LTC4 LTD4 and LTE4), only LTE4 is sufficiently stable to be detectable in extracellular fluids. However, LTE4 has received little attention because it binds poorly to the CysLT1 and CysLT2 receptors; therefore, little is known about the effects of LTE4 on mucous secretion. Recently, studies have focused on the P2Y12 receptor as a potential receptor for LTE4, because this receptor is required for LTE4-mediated pulmonary inflammation. In our previous study, we confirmed the expression of P2Y12 receptor in human airway epithelial cells. To clarify the roles of LTE4 in airway epithelial cells, we investigated mucus secretion by LTE4 in vitro. MethodsConfluent NCI-H292 cells were stimulated with LTE4 (0.01–1 μM) for 24 h. The release and production of MUC5AC protein, a gel-forming mucin, were evaluated with an enzyme-linked immunosorbent assay. ResultsWestern blot analysis revealed that NCI-H292 cells expressed P2Y12 receptor protein. LTE4 significantly induced the release of MUC5AC mucin in a dose-dependent manner. Th2 cytokines such as IL-4 (10 ng/mL) and IL-13 (10 ng/mL) accelerated the LTE4-induced release of MUC5AC protein. MRS2935, a P2Y12 receptor antagonist, partially inhibited the LTE4-induced release of MUC5AC protein in the airway. In contrast, MK571, a CysLT1 receptor antagonist, did not affect the release of MUC5AC protein elicited by LTE4. ConclusionsThese results suggest that LTE4 may play some important roles in allergic mucus secretion partially via activation of P2Y12 receptor.

Effect of Multi-Walled Carbon Nanotubes on MUC5AC and MUC5B Expression in Airway Epithelial Cells

Received March 25, 2015 Revised May 8, 2015 Accepted May 17, 2015 Address for correspondence Yong-Dae Kim, MD, PhD Department of OtorhinolaryngologyHead and Neck Surgery, College of Medicine, Yeungnam University, 170 Hyeonchung-ro, Nam-gu, Daegu 705-703, Korea Tel +82-53-620-3781 Fax +82-53-628-7884 E-mail ydkim@med.yu.ac.kr Background and ObjectivesZZMulti-walled carbon nanotubes (MWCNT) are one of the most commonly used nanomaterials to date. Recent studies have demonstrated that MWCNT increase immune response and allergic inflammation in airway epithelial cells. However, the effects of MWCNT on mucin in human airway epithelial cells have not been reported. Therefore, in the present study, the effect of MWCNT on MUC16, MUC5AC, and MUC5B expressions were investigated in human airway epithelial cells. Subjects and MethodZZIn mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effects of MWCNT on MUC16, MUC5AC, and MUC5B expression were analyzed by reverse transcription polymerase chain reaction, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. ResultsZZIn human NCI-H292 airway epithelial cells, MWCNT significantly induced the expression MUC5AC and MUC5B mRNA and the production of MUC5AC and MUC5B protein. However, MWCNT did not induce the expression of MUC16 mRNA. In the primary cultures of normal nasal epithelial cells, MWCNT also induced the expression of MUC5AC and MUC5B mRNA and the production of MUC5AC and MUC5B proteins. ConclusionZZThe results of this study demonstrate that MWCNT induces MUC5AC and MUC5B expression in human airway epithelial cells. These findings provide important information about the biological role of MWCNT on mucus-secretion in human airway epithelial cells. Korean J Otorhinolaryngol-Head Neck Surg 2015;58(8):552-7

MUC5AC hypomethylation is a predictor of microsatellite instability independently of clinical factors associated with colorectal cancer.

Colorectal cancers (CRC) with microsatellite instability (MSI) display unique clinicopathologic features including a mucinous pattern with frequent expression of the secreted mucins MUC2 and MUC5AC. The mechanisms responsible for this altered pattern of expression remain largely unknown. We quantified DNA methylation of mucin genes (MUC2, MUC5AC, MUC4) in colonic cancers and examined the association with clinicopathological characteristics and molecular (MSI, KRAS, BRAF, and TP53 mutations) features. A control cohort was used for validation. We detected frequent hypomethylation of MUC2 and MUC5AC in CRC. MUC2 and MUC5AC hypomethylation was associated with MUC2 and MUC5AC protein expression (p = 0.004 and p < 0.001, respectively), poor differentiation (p = 0.001 and p = 0.007, respectively) and MSI status (p < 0.01 and p < 0.001, respectively). Interestingly, MUC5AC hypomethylation was specific to MSI cancers. Moreover, it was significantly associated with BRAF mutation and CpG island methylator phenotype (p < 0.001 and p < 0.001, respectively). All these results were confirmed in the control cohort. In the multivariate analysis, MUC5AC hypomethylation was a highly predictive biomarker for MSI cancers. MUC5AC demethylation appears to be a hallmark of MSI in CRC. Determination of MUC5AC methylation status may be useful for understanding and predicting the natural history of CRC.

Effects of tumor necrosis factor-α converting enzyme on mucous hypersecretion in inflammatory airway

To investigate the effect of tumor necrosis factor-α converting enzyme (TACE) on mucous hypersecretion in inflammatory airway. Mucous hypersecretion model of human lung adenocarcinoma cells A549 was induced by human neutrophil elastase (HNE), and TNF-α converting enzyme inhibitor-1 (TAPI-1), an inhibitor of TACE, was chosen for the inference study. The expression of MUC5AC and TACE was examined. Th e cells were divided into 5 groups: a negative control group, HNE1 (15 nmol/L) group, HNE2 (25 nmol/L) group, HNE3 (50 nmol/L) group and TAPI-1 group. RT-PCR was used to examine MUC5AC and TACE mRNA expression. The protein expression of TACE and MUC5AC was examined by Western blot and ELISA, respectively. HNE induced the TACE and MUC5AC mRNA and protein expression in a dose-dependent manner. Compared with the control group, the increases were all significantly increased in the three dosages of HNE group (P< 0.01). The HNE-induced TACE and MUC5AC mRNA and protein expression were dramatically attenuated in the presence of TAPI-1, an inhibitor of TACE (P< 0.01). TACE participated cell signalling pathway of airway mucous hypersecretion, and could down regulation the level of inflammation airway mucous hypersecretion.

Apigenin Inhibits Tumor Necrosis Factor-α-Induced Production and Gene Expression of Mucin through Regulating Nuclear Factor-Kappa B Signaling Pathway in Airway Epithelial Cells.

In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-α (TNF-α)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-α for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-κB activation induced by TNF-α. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-κB signaling pathway in airway epithelial cells.

Prognostic significance of CDX2 and mucin expression in small intestinal adenocarcinoma

The clinicopathological and prognostic significance of CDX2 and mucin expression have not been comprehensively evaluated in small intestinal adenocarcinoma. Immunohistochemical microarray analyses of CDX2, MUC1, MUC5AC, and MUC6 protein expressions in 189 surgically resected small intestinal adenocarcinoma cases were examined and compared with various clinicopathologic variables, including survival. CDX2, MUC1, MUC5AC, and MUC6 expressions were observed in 43.4% (82 patients), 37.6% (71), 31.7% (60), and 21.7% (41) of patients, respectively. Whereas CDX2 expression was found to be associated with low-grade tumors (P=0.034), fewer nodal metastases (P=0.019), and less perineural invasion (P=0.049) in small intestinal adenocarcinoma patients, patients expressing MUC1 tended to demonstrate high-grade (P=0.021) and nodular or infiltrative (P=0.020) tumors. On the basis of the combined CDX2, MUC1, MUC5AC, and MUC6 expression patterns, small intestinal adenocarcinoma patients were further classified as intestinal (CDX2+/MUC1−; 29.6%), pancreatobiliary (CDX2−/MUC1+; 23.8%), mixed (CDX2+/MUC1+; 13.8%), gastric (CDX2−/MUC1−/MUC5AC+ or MUC6+; 13.8%), or null (CDX2−/MUC1−/MUC5AC−/MUC6−; 19.0%). Among these immunophenotypes, intestinal-type patients demonstrated more frequent distal (jejunal or ileal; P=0.033), tubular (P=0.039), and low-grade tumors (P=0.004) and significantly better survival according to univariate (P<0.0001) and multivariate (P=0.001) analyses. In summary, intestinal immunophenotype adenocarcinomas are associated with distal (jejunal or ileal), tubular, and low-grade tumors and better survival outcomes. Hence, CDX2 and mucin immunohistochemical staining may provide better estimations of survival after surgical resection and intestinal immunophenotype could therefore be used as a better prognostic indicator of small intestinal adenocarcinoma.

Estradiol increases mucus synthesis in bronchial epithelial cells.

Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.

β2-Adrenoceptor involved in smoking-induced airway mucus hypersecretion through β-arrestin-dependent signaling.

Progression of chronic obstructive pulmonary disease is associated with small airway obstruction by accumulation of inflammatory mucous exudates. However, the mechanism of mucin hypersecretion after exposure to cigarette smoke (CS) is still not clear. In this study, we explored the contribution of β2-adrenoceptor (β2-AR) signaling to CS extract (CSE)-induced mucus hypersecretion in vitro and examined the effect of a β-blocker on airway mucin hypersecretion in vivo. NCI-H292 epithelial cell line was used to determine the contribution of β2-AR signaling to CSE-induced MUC5AC production by treatment with β2-AR antagonists propranolol and ICI118551 and β2-AR-targeted small interfering RNA. The effect of propranolol on airway mucus hypersecretion was examined in a rat model exposed to CS. MUC5AC expression was assayed by real-time PCR, immunohistochemistry and ELISA. β2-AR and its downstream signaling were detected by western blot analysis. We found that pretreating NCI-H292 cells with propranolol, ICI118551 for 30 min or β2AR–targeted siRNA for 48 h reduced MUC5AC mRNA and protein levels stimulated by CSE. However,inhibiting the classical β2AR–cAMP-PKA pathway didn’t attenuate CSE-induced MUC5AC production, while silencing β-arretin2 expression significantly decreased ERK and p38MAPK phosphorylation, thus reduced the CSE-stimulated MUC5AC production. In vivo, we found that administration of propranolol (25 mg kg−1d−1) for 28 days significantly attenuated the airway goblet cell metaplasia, mucus hypersecretion and MUC5AC expression of rats exposed to CS. From the study, β2-AR–β-arrestin2–ERK1/2 signaling was required for CS-induced airway MUC5AC expression. Chronic propranolol administration ameliorated airway mucus hypersecretion and MUC5AC expression in smoking rats. The exploration of these mechanisms may contribute to the optimization of β2-AR target therapy in chronic obstructive pulmonary disease.