Effects of PM2.5 on mucus hypersecretion in airway through miR-133b-5p/EGFR/Claudin1/MUC5AC axis.

OBJECTIVE: To investigate the role of the EGFR/MAPK signaling pathway in PM2.5 in promoting the MUC5AC hypersecretion in airway and exacerbating airway inflammation. METHODS: By establishing rat model exposed to PM2.5, overexpressing miR-133b-5p and Claudin1, the content of IL-1 and TNF-α in serum were detected by ELISA, the pathology of lung tissue was observed by HE staining, p-EGFR, Claudin1, MUC5AC, p-ERK1/2, p-JNK, p-p38 in rats lung tissue were detected by immunohistochemical and WB, the expression level of miR-133b-5p in rats lung tissue were detected by qPCR. RESULTS: After the rats were exposed to PM2.5, the content of inflammatory factors in serum increased, the inflammatory damage of lung tissues occurred, the expression of miR-133b-5p was down-regulated, and the expression of MUC5AC protein was increased. The ELISA test results showed that the expression of IL-1 and TNF-α in the model group was significantly higher than that in the control group, and the model +AG1478 treatment group was down-regulated compared with the model group, and the +miR-133b-5p agomir treatment group was significantly lower than that in the control group, the model group and the model +Claudin1 overexpression blank load group, and the model +Claudin1 overexpression group was down-regulated compared with the model group and the model +Claudin1 overexpression blank load group. The protein detection results showed that the expression of p-EGFR, MUC5AC, p-ERK1/2, p-JNK and p-p38 proteins was increased and the expression of Claudin1 protein was decreased in the model group compared with the control group. In the model + AG1478 treatment group, model + miR-133b-5p agomir treatment group and model + Claudin1 overexpression group, compared with the model group, p-EGFR, MUC5AC, p-ERK1/2, p-JNK, p-p38 protein expression was down-regulated, and Claudin1 protein expression was up-regulated. CONCLUSIONS: PM2.5 inhibited the expression of miR-133b-5p to activate the EGFR/MAPK signal pathway, induce the hypersecretion of MUC5AC, thus aggravating PM2.5-related airway inflammation in rats.

[H3K27 acetylation promotes lncRNA OIP5-AS1 transcription and induces apoptosis of nasal epithelial cells in allergic rhinitis through up-regulation of TLR4].

Objective To investigate the effect of lysine 27 residue of histone H3 (H3K27) acetylation modification on the transcriptional promotion of long noncoding RNA OPA interacting protein 5-antisense RNA 1 (lncRNA OIP5-AS1) and apoptosis of nasal epithelial cells (NECs) in allergic rhinitis (AR) via regulating Toll-like receptor 4 (TLR4). Methods Interleukin-13 (IL-13) was used to treat NECs to establish an AR cell model. Real-time quantitative PCR was utilized to detect the expressions of OIP5-AS1 and TLR4 in nasal mucosal tissues of AR patients and in the in vitro cell model. The concentrations of macrophage colony-stimulating factor (GM-CSF), eotaxin-1, and mucin 5AC (MUC5AC) were detected by ELISA. The apoptosis of NECs was determined by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). A dual-luciferase report experiment was carried out to verify the relationship between OIP5-AS1 and TLR4. Chromatin immunoprecipitation (ChIP) assay was performed to verify H3K27 acetylation of histones in the OIP5-AS1 promoter region. Results Compared with healthy controls and untreated NECs, OIP5-AS1 and TLR4 were both up-regulated in nasal mucosal tissues from AR patients and IL-13-stimulated NECs. Knockdown of OIP5-AS1 decreased the level of TLR4 in IL-13-treated NECs, while overexpression of OIP5-AS1 increased the level of TLR4. Inhibition of OIP5-AS1 reduced the apoptosis rate, and inhibited the secretion of GM-CSF, eotaxin-1, and MUC5AC from IL-13-treated NECs, while overexpression of TLR4 partially reversed the effects of OIP5-AS1 knockdown on NEC apoptosis and the secretion of GM-CSF, eotaxin-1, and MUC5AC. In addition, H3K27 acetylation was markedly enriched in the promoter region of OIP5-AS1, and H3K27 acetylation promoted the expression of OIP5-AS1 in IL-13-treated NECs. Conclusion H3K27 acetylation promotes OIP5-AS1 transcription and induces NEC apoptosis in AR via upregulation of TLR4.

MUC5AC: A potential biomarker of severity in pediatric patients infected with influenza.

Numerous factors can increase the risk of severe influenza; however, a majority of severe cases occur in previously healthy children. Identification of high-risk children is important for targeted preventive interventions and prompt treatment. The aim of this study was to evaluate MUC5AC as a biomarker for influenza disease severity in children. For this, a prospective cohort study was conducted in 2019. Children hospitalized with acute respiratory infection (ARI) with confirmed positive influenza infection were enrolled. Influenza cases were identified by reverse transcriptase-polymerase chain reaction. Life-threatening disease (LTD) was defined by the need for intensive care and ventilatory support. MUC5AC, epidemiologic, and clinical risk factors were assessed. Three hundred and forty-two patients were hospitalized with ARI, of which 49 (14%) had confirmed influenza infection and 6 (12%) of them developed LTD. MUC5AC levels were higher in those patients with mild disease compared to cases with poorer outcomes. Our results show that the severity of influenza infection in children is significantly associated with low levels of MUC5AC. These findings suggest its potential as a suitable biomarker for predicting disease severity.

A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

Seihaito, a Kampo medicine, attenuates IL-13-induced mucus production and goblet cell metaplasia.

Goblet cell hyperplasia and increased mucus production are features of airway diseases, including asthma, and excess airway mucus often worsens these conditions. Even steroids are not uniformly effective in mucus production in severe asthma, and new therapeutic options are needed. Seihaito is a Japanese traditional medicine that is used clinically as an antitussive and expectorant. In the present study, we examined the effect of Seihaito on goblet cell differentiation and mucus production. In in vitro studies, using air-liquid interface culture of guinea-pig tracheal epithelial cells, Seihaito inhibited IL-13-induced proliferation of goblet cells and MUC5AC, a major component of mucus production. Seihaito suppressed goblet cell-specific gene expression, without changing ciliary cell-specific genes, suggesting that it inhibits goblet cell differentiation. In addition, Seihaito suppressed MUC5AC expression in cells transfected with SPDEF, a transcription factor activated by IL-13. Furthermore, Seihaito attenuated in vivo goblet cell proliferation and MUC5AC mRNA expression in IL-13-treated mouse lungs. Collectively, these findings demonstrated that Seihaito has an inhibitory effect on goblet cell differentiation and mucus production, which is at least partly due to the inhibition of SPDEF.

Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.

The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.

Reprogramming of arachidonic acid metabolism using α-terpineol to alleviate asthma: insights from metabolomics.

Asthma is a chronic inflammatory disorder in airways with typical pathologic features of airway inflammation and mucus hypersecretion. α-Terpineol is a monocyclic terpene found in many natural plants and foods. It has been reported to possess a wide range of pharmacological activities including anti-inflammatory and expectorant effects. However, the role of α-terpineol in asthma and its potential protective mechanism have not been well elucidated. This study is designed to investigate the pharmacological effect and mechanism of α-terpineol on asthmatic mice using the metabolomics platform. A murine model of asthma was established using ovalbumin (OVA) sensitization and then challenged for one week. The leukocyte count and inflammatory cytokines in the bronchoalveolar lavage fluid (BALF), lung histopathology, inflammatory  infiltrate and mucus secretion were evaluated. An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based metabolomics study was performed on lung tissues and serum to explore endogenous small molecule metabolites affected by α-terpineol in asthmatic mice. After α-terpineol treatment, leukocyte count, inflammatory cytokines in the BALF, and peribronchial inflammation infiltration were significantly downregulated. Goblet cell hyperplasia and mucus secretion were attenuated, with the level of Muc5ac in BALF decreased. These results proved the protective effect of α-terpineol against airway inflammation, mucus hypersecretion and Th1/Th2 immune imbalance. To further investigate the underlying mechanisms of α-terpineol in asthma treatment, UPLC-MS/MS-based metabolomics analysis was performed. 26 and 15 identified significant differential metabolites were found in the lung tissues and serum of the control, model and α-terpineol groups, respectively. Based on the above differential metabolites, enrichment analysis showed that arachidonic acid (AA) metabolism was reprogrammed in both mouse lung tissues and serum. 5-Lipoxygenase (5-LOX) and cysteinyl leukotrienes (CysLTs) are the key enzyme and the end product of AA metabolism, respectively. In-depth studies have shown that pretreatment with α-terpineol can alleviate asthma by decreasing the AA level, downregulating the expression of 5-LOX and reducing the accumulation of CysLTs in mouse lung tissues. In summary, this study demonstrates that α-terpineol is a potential agent that can prevent asthma via regulating disordered AA metabolism.

Assessment of MUC5AC and MUC2 Immunoexpression in Glandular Odontogenic Cysts, Dentigerous Cysts, and Mucoepidermoid Carcinomas.

Glandular odontogenic cysts (GOCs) and dentigerous cysts may show mucous metaplasia. Central mucoepidermoid carcinoma is very rare and mostly associated with dental cysts. It is hypothesized that odontogenic cysts showing mucus differentiation in their lining, have a propensity to transform into MEC. The present study is the first attempt to explore the relationship between odontogenic cysts [GOCs and dentigerous cysts with mucus metaplasia (DCMM)] and MEC by evaluating immunoexpression of MUC5AC and MUC2. Immunoexpression of MUC5AC and MUC2 was evaluated semiquantitatively in GOCs (20 cases), DCMMs (20 cases), and MECs (20 cases). The percentage of positive cells, intensity, and localization of immunoexpression were assessed for each marker in all cases. Of GOCs, DCMMs, and MECs cases, 85%, 70%, and 80%, respectively, were immunopositive for MUC5AC. Strong cytoplasmic immunoreactivity for MUC5AC was noted, particularly in mucous cells present diffusely within MECs. However, the immunoreactivity was limited to the epithelial lining of GOCs and DCMMs. Most of the MECs (60%) showed more than 25% positivity for MUC5AC, followed by GOCs, and the least in DMMCs. Mild cytoplasmic and nuclear positivity of MUC2 was noted only in epithelial lining cells of 70% GOCs and 45% DCMMs. Whereas, 55% of MECs displayed moderate to strong cytoplasmic and membranous immunopositivity for MUC2 exclusively within mucous cells. As MECs showed strong MUC5AC immunoreactivity in mucous cells, immunoexpression of MUC5AC in odontogenic cysts with mucus cells can possibly explain the pathogenesis of MEC from cysts. However, the variable expression of MUC2 did not give any strong evidence regarding its role as a marker.

MUC5AC concentrations in lung lavage fluids are associated with acute lung injury after cardiac surgery.

Heart surgery may be complicated by acute lung injury and adult respiratory distress syndrome. Expression and release of mucins MUC5AC and MUC5B in the lungs has been reported to be increased in acute lung injury. The aim of our study was to [1] investigate the perioperative changes of MUC5AC, MUC5B and other biomarkers in mini-bronchoalveolar lavage (minBAL), and [2] relate these to clinical outcomes after cardiac surgery. In this prospective cohort study in 49 adult cardiac surgery patients pre- and post-surgery non-fiberscopic miniBAL fluids were analysed for MUC5AC, MUC5B, IL-8, human neutrophil elastase, and neutrophils. All measured biomarkers increased after surgery. Perioperative MUC5AC-change showed a significant negative association with postoperative P/F ratio (p = 0.018), and a positive association with ICU stay (p = 0.027). In conclusion, development of lung injury after cardiac surgery and prolonged ICU stay are associated with an early increase of MUC5AC as detected in mini-BAL.

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα.

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air-liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1ß contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1ß-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1ß-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

Investigation of Conjunctival Goblet Cell and Tear MUC5AC Protein in Patients With Graves' Ophthalmopathy.

Purpose: The aim of this study was to investigate conjunctival goblet cell density (GCD) and tear mucin-5AC (MUC5AC) protein levels in patients with Graves' ophthalmopathy (GO) and their association with dry eye indicators. Methods: A total of 99 patients with GO (54 active, 45 inactive) and 40 healthy controls were recruited. Comprehensive ophthalmic examinations, including the external eye, ocular surface, GCD, and tear MUC5AC ELISA, were performed. The GCD examination was performed in temporal bulbar conjunctiva, including IVCM GCD by in vivo confocal microscopy (IVCM) and filled GCD of cytokeratin-7 and MUC5AC-positive co-immunomarkers by impression cytology. Tear MUC5AC protein was detected using samples extracted from Schirmer strips. Results: The GO group showed a significant decrease in IVCM GCD, filled GCD, and normalized tear MUC5AC protein compared to controls, with the active GO group showing the greatest decrease (all P < 0.05). Tear MUC5AC protein levels in GO correlated with those of IVCM GCD (r = 0.40, P < 0.001) and filled GCD (r = 0.54, P < 0.001, respectively). Higher ocular surface disease index (r = -0.22, P < 0.05; r = -0.20, P < 0.05; r = -0.21, P < 0.05) and lisamine green staining (r = -0.23, P < 0.05; r = -0.38, P < 0.001; r = -0.42, P < 0.001) were associated with lower tear MUC5AC protein levels, IVCM GCD, and filled GCD, respectively, which decreased with increasing clinical activity score (r = -0.24, P < 0.05; r = -0.28, P < 0.01; r = -0.27, P < 0.01) and conjunctival congestion score (r = -0.27, P < 0.01; r = -0.33, P < 0.001; r = -0.42, P < 0.001). Conclusions: The goblet cell count and tear MUC5AC protein in GO eyes were decreased, possibly due to ocular surface inflammation. Translational Relevance: This study observed the change of tear film mucin in GO patients.

The SIRT3 activator ganoderic acid D regulates airway mucin MUC5AC expression via the NRF2/GPX4 pathway.

PURPOSE: The expression of MUC5AC, a highly prevalent airway mucin, is regulated by stimulatory factors such as oxidative stress. Ganoderic acid D (GAD) activates mitochondrial deacetylase SIRT3. SIRT3 regulates mitochondrial function through deacetylation of mitochondrial proteins, thereby playing a significant role in alleviating oxidative stress-related diseases. Therefore, this study aimed to investigate the mechanisms and rationale underlying the regulation of MUC5AC expression by GAD. METHODS: Human airway epithelial cells (NCI-H292) were exposed to pyocyanin (PCN) to establish an in vitro cell model of airway mucus hypersecretion. The expression of SIRT3, MUC5AC, and NRF2 pathway proteins in cells was assessed. Cellular mitochondrial morphology and oxidative stress markers were analyzed. C57BL/6 mice were induced with Pseudomonas aeruginosa (PA) to establish an in vivo mouse model of airway mucus hypersecretion. The expression of SIRT3 and MUC5AC in the airways was examined. In addition, the differential expression of target genes in the airway epithelial tissues of patients with chronic obstructive pulmonary disease (COPD) was analyzed using publicly available databases. RESULTS: The results revealed a significant upregulation of MUC5AC expression and a significant downregulation of SIRT3 expression in relation to airway mucus hypersecretion. GAD inhibited the overexpression of MUC5AC in PCN-induced NCI-H292 cells and PA-induced mouse airways by upregulating SIRT3. GAD activated the NRF2/GPX4 pathway and inhibited PCN-induced oxidative stress and mitochondrial morphological changes in NCI-H292 cells. However, ML385 inhibited the regulatory effects of GAD on MUC5AC expression. CONCLUSION: The SIRT3 activator GAD downregulated MUC5AC expression, potentially through activation of the NRF2/GPX4 pathway. Accordingly, GAD may be a potential treatment approach for airway mucus hypersecretions.

Plasma Bile Acid Profiling and Modulation of Secreted Mucin 5AC in Cholangiocarcinoma.

Studies investigating the potential role of circulating bile acids (BAs) as diagnostic biomarkers for cholangiocarcinoma (CCA) are sparse and existing data do not adjust for confounding variables. Furthermore, the mechanism by which BAs affect the expression of the oncogenic mucin 5AC (MUC5AC) has never been investigated. We performed a case-control study to characterise the profile of circulating BAs in patients with CCA (n = 68) and benign biliary disease (BBD, n = 48) with a validated liquid chromatography-tandem mass spectrometry technique. Odd ratios (OR) for CCA associations were calculated with multivariable logistic regression models based on a directed acyclic graph structure learning algorithm. The most promising BAs were then tested in an in vitro study to investigate their interplay in modulating MUC5AC expression. The total concentration of BAs was markedly higher in patients with CCA compared with BBD controls and accompanied by a shift in BAs profile toward a higher proportion of primary conjugated BAs (OR = 1.50, CI: 1.14 to 1.96, p = 0.003), especially taurochenodeoxycholic acid (TCDCA, OR = 42.29, CI: 3.54 to 504.63, p = 0.003) after multiple adjustments. Western blot analysis of secreted MUC5AC in human primary cholangiocytes treated with primary conjugated BAs or with TCDCA alone allowed us to identify a novel 230 kDa isoform, possibly representing a post-translationally modified MUC5AC specie.

C-C Motif Chemokine Receptor 3-Mediated Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Signaling: Promising Targets for Human Airway Epithelial Mucin 5AC Induction by Eotaxin-2 and Eotaxin-3.

INTRODUCTION: Eotaxin-2 and -3 of the C-C chemokine subfamily function as potent chemoattractant factors for eosinophil recruitment and various immune responses in allergic and inflammatory airway diseases. Mucin 5AC (MUC5AC), a major gel-forming secretory mucin, is overexpressed in airway inflammation. However, the association between mucin secretion and eotaxin-2/3 expression in the upper and lower airway epithelial cells has not been fully elucidated. Therefore, in this study, we investigated the effects of eotaxin-2/3 on MUC5AC expression and its potential signaling mediators. METHODS: We analyzed the effects of eotaxin-2 and -3 on NCI-H292 human airway epithelial cells and primary human nasal epithelial cells (HNEpCs) via reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. Along with immunoblot analyses with specific inhibitors and small interfering RNA (siRNA), we explored the signaling pathway involved in MUC5AC expression following eotaxin-2/3 treatment. RESULTS: In HCI-H292 cells, eotaxin-2/3 activated the mRNA expression and protein production of MUC5AC. A specific inhibitor of C-C motif chemokine receptor 3 (CCR3), SB328437, suppressed eotaxin-2/3-induced MUC5AC expression at both the mRNA and protein levels. Eotaxin-2/3 induced the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38, whereas pretreatment with a CCR3 inhibitor significantly attenuated this effect. Induction of MUC5AC expression with eotaxin-2/3 was decreased by U0126 and SB203580, specific inhibitors of ERK1/2 and p38 mitogen-activated protein kinase (MAPK), respectively. In addition, cell transfection with ERK1/2 and p38 siRNAs inhibited eotaxin-2/3-induced MUC5AC expression. Moreover, specific inhibitors (SB328437, U0126, and SB203580) attenuated eotaxin-2/3-induced MUC5AC expression in HNEpCs. CONCLUSION: Our results imply that CCR3-mediated ERK1/2 and p38 MAPK are involved in the signal transduction of eotaxin-2/3-induced MUC5AC overexpression.

ANRIL regulating the secretion of Muc5ac induced by atmospheric PM2.5 via NF-κB pathway in Beas-2B cells.

PM2.5 can cause airway inflammation and promote the excessive secretion of mucin 5ac (Muc5ac), which can further induce many respiratory diseases. Antisense non-coding RNA in the INK4 locus (ANRIL) might regulate the inflammatory responses mediated by nuclear factor kappa-B (NF-κB) signaling pathway. Beas-2B cells were used to clarify the role of ANRIL in the secretion of Muc5ac induced by PM2.5 . The siRNA was used to silence ANRIL expression. Normal and gene silenced Beas-2B cells were respectively exposed to different doses of PM2.5 for 6, 12, and 24 h. The survival rate of Beas-2B cells was detected by methyl thiazolyl tetrazolium (MTT) assay. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and Muc5ac levels were determined by enzyme linked immunosorbent assay (ELISA). The expression levels of NF-κB family genes and ANRIL were detected by real time polymerase chain reaction (PCR). The levels of NF-κB family proteins and NF-κB family phosphorylated proteins were determined using Western blot. Immunofluorescence experiments were performed to observe the nuclear transposition of RelA. PM2.5 exposure increased the levels of Muc5ac, IL-1ß and TNF-α, and ANRIL gene expression (p < .05). With the dose and time of PM2.5 exposure increasing, the protein levels of inhibitory subunit of nuclear factor kappa-B alpha (IκB-α), RelA, and NF-κB1 decreased, the protein levels of phosphorylated RelA (p-RelA) and phosphorylated NF-κB1 (p-NF-κB1) increased, and RelA nuclear translocation increased, which indicated that the NF-κB signaling pathway was activated (p < .05). Silencing ANRIL could decrease the levels of Muc5ac, IL-1ß, TNF-α, decrease NF-κB family genes expression, inhibit the degradation of IκB-α and the activation of NF-κB pathway (p < .05). ANRIL played a regulatory role in the secretion of Muc5ac and the inflammation induced by atmospheric PM2.5 via NF-κB pathway in Beas-2B cells. ANRIL could be a target for prevention and treatment of the respiratory diseases caused by PM2.5 .

Immunohistochemical detection of MUC5AC and MUC5B mucins in ferrets.

OBJECTIVE: Cystic fibrosis (CF) is a genetic condition that causes abnormal mucus secretions in affected organs. MUC5AC and MUC5B are gel-forming mucins and frequent targets for investigations in CF tissues. Our objective was to qualify MUC5AC and MUC5B immunohistochemical techniques to provide a useful tool to identify, localize and interpret mucin expression in ferret tissues. RESULTS: MUC5AC and MUC5B mucins were detected most commonly in large airways and least in small airways, consistent with reported goblet cell density in airway surface epithelia. We evaluated whether staining method affected the detection of goblet cell mucins in serial sections of bronchial surface epithelia. Significant differences between stains were not observed suggesting common co-expression MUC5AC and MUC5B proteins in goblet cells of airway surface epithelia. Gallbladder and stomach tissues are reported to have differential mucin enrichment, so we tested these tissues in wildtype ferrets. Stomach tissues were enriched in MUC5AC and gallbladder tissues enriched in MUC5B, mucin enrichment similar to human tissues. Mucin immunostaining techniques were further qualified for specificity using lung tissue from recently generated MUC5AC-/- and MUC5B-/- ferrets. Qualified techniques for MUC5AC and MUC5B immunohistochemistry will be useful tools for mucin tissue studies in CF and other ferret models.

Alterations in p53, Microsatellite Stability and Lack of MUC5AC Expression as Molecular Features of Colorectal Carcinoma Associated with Inflammatory Bowel Disease.

Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the "chronic inflammation-dysplasia-cancer" carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations.

Predictive Value of MUC5AC Signature in Pancreatic Ductal Adenocarcinoma: A Hypothesis Based on Preclinical Evidence.

Mucin 5AC (MUC5AC) glycoprotein plays a crucial role in carcinogenesis and drug sensitivity in pancreatic ductal adenocarcinoma (PDAC), both individually and in combination with other mucins. Its function and localization are glycoform-specific. The immature isoform (detected by the CLH2 monoclonal antibody, or mab) is usually in the perinuclear (cytoplasmic) region, while the mature (45 M1, 2-11, Nd2) variants are in apical and extracellular regions. There is preclinical evidence suggesting that mature MUC5AC has prognostic and predictive (response to treatment) value. However, these findings were not validated in clinical studies. We propose a MUC5AC signature with three components of MUC5AC-localization, variant composition, and intensity-suggesting a reliable marker in combination of variants than with individual MUC5AC variants alone. We also postulate a theory to explain the occurrence of different MUC5AC variants in abnormal pancreatic lesions (benign, precancerous, and cancerous). We also analyzed the effect of mature MUC5AC on sensitivity to drugs often used in PDAC management, such as gemcitabine, 5-fluorouracil, oxaliplatin, irinotecan, cisplatin, and paclitaxel. We found preliminary evidence of its predictive value, but there is a need for large-scale studies to validate them.