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.

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.

Assembly and organization of the N-terminal region of mucin MUC5AC: Indications for structural and functional distinction from MUC5B.

Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, "tethered" mucus layer in chronic muco-obstructive lung diseases.

Mature MUC5AC Expression in Resected Pancreatic Ductal Adenocarcinoma Predicts Treatment Response and Outcomes.

Neoadjuvant therapy (NAT) for early-stage pancreatic ductal adenocarcinoma (PDA) has recently gained prominence. We investigated the clinical significance of mucin 5 AC (MUC5AC), which exists in two major glycoforms, a less-glycosylated immature isoform (IM) and a heavily glycosylated mature isoform (MM), as a biomarker in resected PDA. Immunohistochemistry was performed on 100 resected PDAs to evaluate the expression of the IM and MM of MUC5AC using their respective monoclonal antibodies, CLH2 (NBP2-44455) and 45M1 (ab3649). MUC5AC localization (cytoplasmic, apical, and extra-cellular (EC)) was determined, and the H-scores were calculated. Univariate and multivariate (MVA) Cox regression models were used to estimate progression-free survival (PFS) and overall survival (OS). Of 100 resected PDA patients, 43 received NAT, and 57 were treatment-naïve with upfront surgery (UpS). In the study population (n = 100), IM expression (H-scores for objective response vs. no response vs. UpS = 104 vs. 152 vs. 163, p = 0.01) and MM-MUC5AC detection rates (56% vs. 63% vs. 82%, p = 0.02) were significantly different. In the NAT group, MM-MUC5AC-negative patients had significantly better PFS according to the MVA (Hazard Ratio: 0.2, 95% CI: 0.059-0.766, p = 0.01). Similar results were noted in a FOLFIRINOX sub-group (n = 36). We established an association of MUC5AC expression with treatment response and outcomes.

MUC5AC Genetic Variation Is Associated With Tuberculous Meningitis Cerebral Spinal Fluid Cytokine Responses and Mortality.

BACKGROUND: The purpose of this study was to assess if single nucleotide polymorphisms (SNPs) in lung mucins MUC5B and MUC5AC are associated with Mycobacterium tuberculosis outcomes. METHODS: Independent SNPs in MUC5B and MUC5AC (genotyped by Illumina HumanOmniExpress array) were assessed for associations with tumor necrosis factor (TNF) concentrations (measured by immunoassay) in cerebral spinal fluid (CSF) from tuberculous meningitis (TBM) patients. SNPs associated with CSF TNF concentrations were carried forward for analyses of pulmonary and meningeal tuberculosis susceptibility and TBM mortality. RESULTS: MUC5AC SNP rs28737416 T allele was associated with lower CSF concentrations of TNF (P = 1.8 × 10-8) and IFN-γ (P = 2.3 × 10-6). In an additive genetic model, rs28737416 T/T genotype was associated with higher susceptibility to TBM (odds ratio [OR], 1.24; 95% confidence interval [CI], 1.03-1.49; P = .02), but not pulmonary tuberculosis (OR, 1.11, 95% CI, .98-1.25; P = .10). TBM mortality was higher among participants with the rs28737416 T/T and T/C genotypes (35/119, 30.4%) versus the C/C genotype (11/89, 12.4%; log-rank P = .005) in a Vietnam discovery cohort (n = 210), an independent Vietnam validation cohort (n = 87; 9/87, 19.1% vs 1/20, 2.5%; log-rank P = .02), and an Indonesia validation cohort (n = 468, 127/287, 44.3% vs 65/181, 35.9%; log-rank P = .06). CONCLUSIONS: MUC5AC variants may contribute to immune changes that influence TBM outcomes.

Context-Dependent Roles of Hes1 in the Adult Pancreas and Pancreatic Tumor Formation.

BACKGROUND & AIMS: The Notch signaling pathway is an important pathway in the adult pancreas and in pancreatic ductal adenocarcinoma (PDAC), with hairy and enhancer of split-1 (HES1) as the core molecule in this pathway. However, the roles of HES1 in the adult pancreas and PDAC formation remain controversial. METHODS: We used genetically engineered dual-recombinase mouse models for inducing Hes1 deletion under various conditions. RESULTS: The loss of Hes1 expression in the adult pancreas did not induce phenotypic alterations. However, regeneration was impaired after caerulein-induced acute pancreatitis. In a pancreatic intraepithelial neoplasia (PanIN) mouse model, PanINs rarely formed when Hes1 deletion preceded PanIN formation, whereas more PanINs were formed when Hes1 deletion succeeded PanIN formation. In a PDAC mouse model, PDAC formation was also enhanced by Hes1 deletion after PanIN/PDAC development; therefore, Hes1 promotes PanIN initiation but inhibits PanIN/PDAC progression. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction revealed that Hes1 deletion enhanced epithelial-to-mesenchymal transition via Muc5ac up-regulation in PDAC progression. The results indicated that HES1 is not required for maintaining the adult pancreas under normal conditions, but is important for regeneration during recovery from pancreatitis; moreover, Hes1 plays different roles, depending on the tumor condition. CONCLUSIONS: Our findings highlight the context-dependent roles of HES1 in the adult pancreas and pancreatic cancer.

Mucin 5AC-Mediated CD44/ITGB1 Clustering Mobilizes Adipose-Derived Mesenchymal Stem Cells to Modulate Pancreatic Cancer Stromal Heterogeneity.

BACKGROUND & AIMS: Secreted mucin 5AC (MUC5AC) promotes pancreatic cancer (PC) progression and chemoresistance, suggesting its clinical association with poor prognosis. RNA sequencing analysis from the autochthonous pancreatic tumors showed a significant stromal alteration on genetic ablation of Muc5ac. Previously, depletion or targeting the stromal fibroblasts showed an ambiguous effect on PC pathogenesis. Hence, identifying the molecular players and mechanisms driving fibroblast heterogeneity is critical for improved clinical outcomes. METHODS: Autochthonous murine models of PC (KrasG12D, Pdx1-Cre [KC] and KrasG12D, Pdx1-Cre, Muc5ac-/- [KCM]) and co-implanted allografts of murine PC cell lines (Muc5ac wild-type and CRISPR/Cas knockout) with adipose-derived mesenchymal stem cells (AD-MSCs) were used to assess the role of Muc5ac in stromal heterogeneity. Proliferation, migration, and surface expression of cell-adhesion markers on AD-MSCs were measured using live-cell imaging and flow cytometry. MUC5AC-interactome was investigated using mass-spectrometry and enzyme-linked immunosorbent assay. RESULTS: The KCM tumors showed a significant decrease in the expression of α-smooth muscle actin and fibronectin compared with histology-matched KC tumors. Our study showed that MUC5AC, carrying tumor secretome, gets enriched in the adipose tissues of tumor-bearing mice and patients with PC, promoting CD44/CD29 (integrin-ß1) clustering that leads to Rac1 activation and migration of AD-MSCs. Furthermore, treatment with KC-derived serum enhanced proliferation and migration of AD-MSCs, which was abolished on Muc5ac-depletion or pharmacologic inhibition of CXCR2 and Rac1, respectively. The AD-MSCs significantly contribute toward α-smooth muscle actin-positive cancer-associated fibroblasts population in Muc5ac-dependent manner, as suggested by autochthonous tumors, co-implantation xenografts, and patient tumors. CONCLUSION: MUC5AC, secreted during PC progression, enriches in adipose and enhances the mobilization of AD-MSCs. On recruitment to pancreatic tumors, AD-MSCs proliferate and contribute towards stromal heterogeneity.

Experimental study of the mechanism of induction of conjunctival goblet cell hyperexpression using CHIR-99021 in vitro.

A component of the tear film, mucin is produced by conjunctival goblet cells and is crucial to preserving the tear film's stability. Severe thermal burns, chemical burns, and severe ocular surface diseases can cause extensive damage to the conjunctiva, destroy the secretory function of goblet cells, and affect the stability of the tear film and integrity of the ocular surface. Currently, the expansion efficiency of goblet cells in vitro is low. In this study, we observed that rabbit conjunctival epithelial cells exhibited dense colony morphology after stimulation with the Wnt/ß-catenin signaling pathway activator CHIR-99021 and promoted the differentiation of conjunctival goblet cells and the expression of its specific marker Muc5ac, among which the best induction effect was observed after 72 h in vitro culture with 5 µmol/L CHIR-99021. Under optimal culture conditions, CHIR-99021 increased the expression levels of the Wnt/ß-catenin signaling pathway factors Frzb, ß-catenin, SAM pointed domain containing ETS transcription factor, and glycogen synthase kinase-3ß and the levels of the Notch signaling pathway factors Notch1 and Krüppel-like factor 4 while decreasing the expression levels of Jagged-1 and Hes1. The expression level of ABCG2, a marker of epithelial stem cells, was raised to keep rabbit conjunctival epithelial cells from self-renewing. Our study showed that CHIR-99021 stimulation successfully activated the Wnt/ß-catenin signaling pathway and conjunctival goblet cell differentiation was stimulated, in which the Notch signaling pathway played a combined role. Those results provide a novel idea for the expansion of goblet cells in vitro.

Proteome of airway surface liquid and mucus in newborn wildtype and cystic fibrosis piglets.

BACKGROUND: The respiratory tract is protected from inhaled particles and microbes by mucociliary clearance, mediated by the mucus and the cilia creating a flow to move the mucus cephalad. Submucosal glands secrete linear MUC5B mucin polymers and because they pass through the gland duct before reaching the airway surface, bundled strands of 1000-5000 parallel molecules exit the glands. In contrast, the surface goblet cells secrete both MUC5AC and MUC5B. METHODS: We used mass-spectrometry based proteomic analysis of unstimulated and carbachol stimulated newborn wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) null (CF) piglet airways to study proteins in the airway surface liquid and mucus, to investigate if levels of MUC5AC and MUC5B were affected by carbachol stimulation and whether the proteins clustered according to function. RESULTS: Proteins in the first four extracted fractions clustered together and the fifth fraction contained the mucus cluster, mucins and other proteins known to associate with mucins, whereas the traditional airway surface liquid proteins clustered to fraction 1-4 and were absent from the mucus fraction. Carbachol stimulation resulted in increased MUC5AC and MUC5B. CONCLUSIONS: These results indicate a distinct separation between proteins in the washable surface liquid and the mucus fraction. In fractions 1-4 from newborn CF piglets an additional cluster containing acute phase proteins was observed, suggesting an early inflammatory response in CF piglets. Alternatively, increased levels of these proteins could indicate altered lung development in the CF piglets. This observation suggests that CF airway disease is present at birth and thus, treatment should commence directly after diagnosis.

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.

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.

House dust mite-induced endoplasmic reticulum stress mediates MUC5AC hypersecretion via TBK1 in airway epithelium.

Purpose: Endoplasmic reticulum (ER) stress regulates mucus hypersecretion, and may activate downstream factors via TBK1 signaling to induce gene expression. However, it remains unclear whether ER stress promotes airway mucus secretion through the TBK1 pathway. We aimed to investigate the role of the TBK1 pathway in the regulation of MUC5AC expression in a mouse model of house dust mite (HDM)-induced allergic asthma. Materials and Methods: Mice with HDM-induced asthma and human bronchial epithelial BEAS-2B cells were treated with amlexanox, an anti-allergy drug (25 µM), or 4-PBA (10 mM). Tissue and cell samples were collected. Tissue samples were stained with hematoxylin and eosin (H&E) or periodic acid Schiff (PAS) to evaluate pathology. Protein expression was analyzed by western blotting and immunofluorescence. Results: Mice exposed to HDM presented ER stress and hypersecretion of mucus Muc5ac from airway epithelial cells (p < 0.001). Similar results were observed in BEAS-2B cells following exposure to HDM. Both in vivo and in vitro studies revealed that HDM-induced ER stress induced MUC5AC overexpression via TBK1 signaling. Amlexanox and 4-PBA markedly reduced mucus production and weakened the TBK1 signal, which mediates MUC5AC hypersecretion. Conclusion: TBK1 plays a pivotal role in HDM-induced ER stress, leading to overproduction of MUC5AC in the asthmatic airway epithelium. The overproduction of MUC5AC can be significantly decreased by inhibiting TBK1 or ER stress using 4-PBA. These findings highlight potential target-specific therapies for patients with chronic allergic asthma.

Genetic analyses of chr11p15.5 region identify MUC5AC-MUC5B associated with asthma-related phenotypes.

OBJECTIVE: Genome-wide association studies (GWASs) have identified single nucleotide polymorphisms (SNPs) in chr11p15.5 region associated with asthma and idiopathic interstitial pneumonias (IIPs). We sought to identify functional genes for asthma by combining SNPs and mRNA expression in bronchial epithelial cells (BEC) in the Severe Asthma Research Program (SARP). METHODS: Correlation analyses of mRNA expression of six candidate genes (AP2A2, MUC6, MUC2, MUC5AC, MUC5B, and TOLLIP) and asthma phenotypes were performed in the longitudinal cohort (n = 156) with RNAseq in BEC, and replicated in the cross-sectional cohort (n = 155). eQTL (n = 114) and genetic association analysis of asthma severity (426 severe vs. 531 non-severe asthma) were performed, and compared with previously published GWASs of IIPs and asthma. RESULTS: Higher expression of AP2A2 and MUC5AC and lower expression of MUC5B in BEC were correlated with asthma, asthma exacerbations, and T2 biomarkers (P < 0.01). SNPs associated with asthma and IIPs in previous GWASs were eQTL SNPs for MUC5AC, MUC5B, or TOLLIP, however, they were not in strong linkage disequilibrium. The risk alleles for asthma or protective alleles for IIPs were associated with higher expression of MUC5AC and lower expression of MUC5B. rs11603634, rs12788104, and rs28415845 associated with moderate-to-severe asthma or adult onset asthma in previous GWASs were not associated with asthma severity (P > 0.8). CONCLUSIONS: SNPs associated with asthma in chr11p15.5 region are not associated with asthma severity neither with IIPs. Higher expression of MUC5AC and lower expression of MUC5B are risk for asthma but protective for IIPs.

Exogenous Lipoxin A4 attenuates IL4-induced Mucin Expression in Human Airway Epithelial Cells.

Introduction: The proinflammatory cytokine interleukin-4 (IL-4) induces mucus hypersecretion by human airway epithelial cells and the MAP kinase signalling pathway may be important in terms of IL-4-induced MUC5AC gene expression. Lipoxin A4 (LXA4) is an arachidonic acid-derived mediator that promotes inflammation by binding to the anti-inflammatory receptors (ALXs) or the formyl-peptide receptor like-1 (FPRL1) protein expressed by airway epithelial cells. Here, we explore the effects of LXA4 on IL-4-induced mucin gene expression in, and secretion from, human airway epithelial cells. Methods: We co-treated cells with IL-4 (20 ng/mL) and LXA4 (1 nM) and measured the expression levels of mRNAs encoding MUC5AC and 5B via real-time polymerase chain reaction; protein expression levels were determined by Western blotting and immunocytofluorescence. The ability of IL-4 and LXA4 to suppress protein expression was determined by Western blotting. Results: IL-4 increased MUC5AC and 5B gene and protein expression. LXA4 suppressed IL-4-induced MUC5AC and 5B gene and protein expression by interacting with the IL4 receptor and mitogen-activated protein kinase (MAPK) pathway, including both phospho-p38 MAPK and phospho-extracellular signal-regulated kinase (phospho-ERK). IL-4 and LXA4 increased and decreased, respectively, the number of cells that stained with anti-MUC5AC and 5B antibodies. Conclusions: LXA4 may regulate mucus hypersecretion induced by IL4 in human airway epithelial cells.

HDAC3-mediated lncRNA ZFAS1 inhibited IL-13-induced secretion of proinflammatory cytokines in nasal epithelial cells by regulating the miR-7-5p/SIRT1 pathway.

Allergic rhinitis (AR) is a disease that is difficult to cure and accompanies the patient's life. Proinflammatory cytokines (GM-CSF and eotaxin) and MUC5AC are key mediators promoting AR progression. Herein, the function of lncRNA ZFAS1 in AR was investigated. Nasal epithelial cells (NECs) were subjected to 50 ng/mL IL-13 for 24 h to construct an AR cell model. The mRNA and protein expressions were assessed using qRT-PCR and western blot. The levels of GM-CSF, eotaxin, IL-1ß, IL-6, TNF-α and MUC5AC in cell supernatant were examined by ELISA. The binding relationships between HDAC3, ZFAS1, miR-7-5p and SIRT1 were analysed using dual luciferase reporter or ChIP assays. Herein, our results displayed that ZFAS1 and SIRT1 were lowly expressed in AR, while miR-7-5p and HDAC3 were highly expressed. Functional experiments displayed that ZFAS1 overexpression suppressed IL-13-induced proinflammatory cytokines and mucin production in NECs. The highly expressed HDAC3 in AR inhibited ZFAS1 expression by binding with ZFAS1 promoter. In addition, our experiments revealed that ZFAS1 targeted miR-7-5p, and miR-7-5p targeted SIRT1. As expected, miR-7-5p overexpression or SIRT1 silencing abrogated ZFAS1 upregulation's repression on IL-13-induced proinflammatory cytokines and MUC5AC secretory levels in NECs. ZFAS1 suppressed proinflammatory cytokines, inflammatory cytokines, and MUC5AC secretory levels in AR by regulating the miR-7-5p/SIRT1 axis. Thus, our work suggested that ZFAS1 might serve as a novel target for AR treatment and prevention.

Molecular insight of p53/Sp1/MUC5AC axis in the tumorigenesis and progression of lung adenocarcinoma.

The aberrant expression of secretory mucin MUC5AC has been documented during the tumourigenesis and progression of various cancers. However, little is currently known on the function of MUC5AC in lung adenocarcinoma. The present study focused on the tumour-promoting role of MUC5AC and its regulatory mechanisms in lung adenocarcinoma. Firstly, MUC5AC expression was evaluated in NSCLC tissue microarrays by immunohistochemistry. Kaplan-Meier analysis were used to clarify the prognostic value of MUC5AC. Subsequently, small interfering RNA and small hairpin RNA were used to knockdown MUC5AC in lung ADC cell lines to elucidate its role in tumorigenesis and progression of lung adenocarcinoma via in vitro functional assays and xenograft mouse models. Finally, the regulatory mechanisms underlying p53/Sp1/MUC5AC axis were identified through dual-luciferase report. We found that MUC5AC was upregulated in lung ADC tissues and cell lines, especially in KRAS-mutant cases and correlated with poor prognosis. MUC5AC gene silencing resulted in reduced cell proliferation, invasion and migration. Furthermore, knockdown of MUC5AC led to reversion of the epithelial-mesenchymal transition. Additionally, downregulation of MUC5AC reduced tumourigenesis in mouse models. Finally, we found an antagonistic role between Sp1 and p53 in the regulation of MUC5AC gene expression. Our findings suggest that high MUC5AC expression promotes tumourigenesis and progression of lung ADC. Both p53 gene inactivation and Sp1 overexpression in lung ADC may enhance MUC5AC expression, especially in KRAS-mutated cases. Given the paucity of efficient drug-targeted approaches of KRAS-driven lung ADCs, therapies directed at downstream effectors such as MUC5AC could have huge prospects.

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/ß) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1ß and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

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 .

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.