Effect of Korean Red Ginseng and Rg3 on Asian Sand Dust-Induced MUC5AC, MUC5B, and MUC8 Expression in Bronchial Epithelial Cells.

Korean Red ginseng (KRG), commonly used in traditional medicine, has anti-inflammatory, anti- oxidative, and anti-tumorigenic properties. Asian sand dust (ASD) is known to aggravate upper and lower airway inflammatory responses. BEAS-2B cells were exposed to ASD with or without KRG or ginsenoside Rg3. Mucin 5AC (MUC5AC), MUC5B, and MUC8 mRNA and protein expression levels were determined using quantitative RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor kappa B (NF-κB), activator protein 1, and mitogen-activated protein kinase expression and activity were determined using western blot analysis. ASD induced MUC5AC, MUC5B, and MUC8 mRNA and protein expression in BEAS-2B cells, which was significantly inhibited by KRG and Rg3. Although ASD-induced mucin expression was associated with NF-κB and p38 mitogen-activated protein kinase (MAPK) activity, KRG and Rg3 significantly suppressed only ASD-induced NF-κB expression and activity. KRG and Rg3 inhibited ASD-induced mucin gene expression and protein production from bronchial epithelial cells. These results suggest that KRG and Rg3 have potential for treating mucus-producing airway inflammatory diseases.

Caged Dexamethasone/Quercetin Nanoparticles, Formed of the Morphogenetic Active Inorganic Polyphosphate, are Strong Inducers of MUC5AC.

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.

Molecular mechanism underlying miR­130b­Sp1 transcriptional regulation in LPS­induced upregulation of MUC5AC in the bile duct epithelium.

Hepatolithiasis is a common disease that represents a serious health threat to the Chinese population. The pathological mechanism underlying hepatolithiasis is closely related to bacterial infections of the intrahepatic bile duct, followed by chronic inflammation and the overexpression of mucin 5AC (MUC5AC). However, the exact mechanism responsible for the lipopolysaccharide (LPS)­induced upregulation of MUC5AC has yet to be elucidated. Specificity protein 1 (Sp1) is a ubiquitous transcription factor that plays a vital role in the regulation of a number of genes that are responsible for normal cellular function. microRNA (miR/miRNA)­130b is a member of the miRNA family. miRNAs can bind to the 3'­untralsated region (3'­UTR) of a target gene and influence its expression levels. The present study found that LPS increases the expression of MUC5AC by influencing Sp1 secretion. Chromatin immunoprecipitation­quantitative PCR experiments further verified three Sp1 binding sites in the MUC5AC promoter sequence that can regulate the expression of MUC5AC. Further analysis demonstrated that Sp1 expression was regulated by miR­130b. Luciferase experiments identified one miR­130b binding site in the Sp1 3'­UTR region. In vivo experiments also confirmed the role of the miR­130b­Sp1­MUC5AC signaling pathway in the formation of biliary stones and indicated that this pathway may provide targeted therapeutic strategies for the treatment of intrahepatic bile duct stones.

Assessment of mucin-related gene alterations following treatment with rebamipide ophthalmic suspension in Sjögren's syndrome-associated dry eyes.

Ocular surface mucins are thought to play vital roles in maintaining the homeostasis of the pre-ocular surface tear film. We performed ocular surface tests with impression cytology to assess the expression levels of mucin-related genes on the ocular surface in healthy eyes. In addition, we investigated alterations in mucin-related gene expression secondary to treatment with rebamipide ophthalmic suspension in patients with Sjögren's syndrome-associated dry eyes (SS-DE). Thirty-three healthy individuals (control group) and 13 patients from our hospital with SS-DE were enrolled. Impression cytology was performed using Schirmer's test paper for RNA sampling. The mRNA levels of SAM-pointed domain-containing ETS-like factor (SPDEF), mucin 5AC (MUC5AC), and mucin 16 (MUC16) were determined using a real-time reverse transcription-polymerase chain reaction. The ocular surface test was performed once for the control group, and at baseline as well as 2, 4, 8, and 12 weeks after treatment in the Sjögren's syndrome-associated dry eyes group. mRNA levels of SPDEF, MUC5AC, and MUC16 were not significantly different between the control and SS-DE groups before rebamipide ophthalmic suspension treatment. SPDEF mRNA levels in control subjects were significantly correlated with levels of MUC5AC. Among SS-DE patients, SPDEF mRNA levels were significantly increased at 2, 4, and 8 weeks after treatment compared with baseline levels. MUC16 mRNA levels were significantly decreased from baseline levels at 4 and 8 weeks post-treatment. Ocular surface test using impression cytology is a clinically useful tool for assessing mucous conditions on the ocular surface and can be used to determine the effects of instillation treatment with eye drops that affect mucin production at the ocular surface.

The effect of Notch signal pathway on PM2.5-induced Muc5ac in Beas-2B cells.

BACKGROUND: Atmospheric pollutants could induced over-expression of Muc5ac, which is a major pathological feature in acute exacerbation of Chronic Obstructive Pulmonary Disease (COPD) and fatal asthma. Notch signaling pathway could promote mucus cell proliferation and mucus secretion. However, the effects of Notch signaling pathway on the airway mucus secretion induced by PM2.5 remain unknown. In this study, we investigated the role of the Notch signaling pathway on Muc5ac by atmospheric PM2.5 in Beas-2B cell. METHODS: The mRNA and protein levels of the Notch1-4, downstream target gene Hes1 and Muc5ac in the Notch signaling pathway were detected by qPCR and western after Beas-2B cells were exposed to PM2.5 of different concentrations for 12h, 24h, and 48h. RESULTS: The longer the exposure time and the higher the concentration of PM2.5, the lower the survival rate of Beas-2B cells. The expressions of Hes1 and Muc5ac in mRNA and protein were significantly increased after PM2.5 exposure. Correlation analysis indicated that there was a positive correlation between the expression of Muc5ac and Hes1 in mRNA and protein. CONCLUSION: Atmospheric PM2.5 can induce the express of Muc5ac, the Notch signaling pathway may be involved in the regulation of Muc5ac by Hes1.

Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.

TMEM16A chloride channel does not drive mucus production.

Airway mucus obstruction is the main cause of morbidity in cystic fibrosis, a disease caused by mutations in the CFTR Cl- channel. Activation of non-CFTR Cl- channels such as TMEM16A can likely compensate for defective CFTR. However, TMEM16A was recently described as a key driver in mucus production/secretion. Here, we have examined whether indeed there is a causal relationship between TMEM16A and MUC5AC production, the main component of respiratory mucus. Our data show that TMEM16A and MUC5AC are inversely correlated during differentiation of human airway cells. Furthermore, we show for the first time that the IL-4-induced TMEM16A up-regulation is proliferation-dependent, which is supported by the correlation found between TMEM16A and Ki-67 proliferation marker during wound healing. Consistently, the notch signaling activator DLL4 increases MUC5AC levels without inducing changes neither in TMEM16A nor in Ki-67 expression. Moreover, TMEM16A inhibition decreased airway surface liquid height. Altogether, our findings demonstrate that up-regulation of TMEM16A and MUC5AC is only circumstantial under cell proliferation, but with no causal relationship between them. Thus, although essential for airway hydration, TMEM16A is not required for MUC5AC production.

Primary infection by Pneumocystis induces Notch-independent Clara cell mucin production in rat distal airways.

Clara cells are the main airway secretory cells able to regenerate epithelium in the distal airways through transdifferentiating into goblet cells, a process under negative regulation of the Notch pathway. Pneumocystis is a highly prevalent fungus in humans occurring between 2 and 5 months of age, a period when airways are still developing and respiratory morbidity typically increases. Pneumocystis induces mucus hyperproduction in immunocompetent host airways and whether it can stimulate Clara cells is unknown. Markers of Clara cell secretion and Notch1 activation were investigated in lungs of immunocompetent rats at 40, 60, and 80 days of age during Pneumocystis primary infection with and without Valproic acid (VPA), a Notch inducer. The proportion of rats expressing mucin increased in Pneumocystis-infected rats respect to controls at 60 and 80 days of age. Frequency of distal airways Clara cells was maintained while mRNA levels for the mucin-encoding genes Muc5B and Muc5ac in lung homogenates increased 1.9 and 3.9 times at 60 days of infection (P. = 0.1609 and P. = 0.0001, respectively) and protein levels of the Clara cell marker CC10 decreased in the Pneumocystis-infected rats at 60 and 80 days of age (P. = 0.0118 & P. = 0.0388). CC10 and Muc5b co-localized in distal airway epithelium of Pneumocystis-infected rats at day 60. Co-localization of Muc5b and Ki67 as marker of mitosis in distal airways was not observed suggesting that Muc5b production by Clara cells was independent of mitosis. Notch levels remained similar and no transnucleation of activated Notch associated to Pneumocystis infection was detected. Unexpectedly, mucus was greatly increased at day 80 in Pneumocystis-infected rats receiving VPA suggesting that a Notch-independent mechanism was triggered. Overall, data suggests a Clara to goblet cell transdifferentiation mechanism induced by Pneumocystis and independent of Notch.

Natural inhibitors on airway mucin: Molecular insight into the therapeutic potential targeting MUC5AC expression and production.

Airway mucin overproduction is the hallmark risk factor of asthma, which is associated with the reduction of lung function. An aberrant mucin expression is responsible for airway obstruction due to its high viscous characteristics. Among the mucins discovered, MUC5AC is the prime mucin of airway epithelia. Nowadays, mucins induced asthma and chronic obstructive pulmonary disease (COPD) are a great concern all over the world. This review focuses on the effects of natural compounds that can be beneficial to explore new drugs to halt MUC5AC secretion and production in airway epithelial, and also their underlying molecular mechanisms based on recent studies. Several researchers are seeking natural sources to identify a new potent MUC5AC inhibitory agent for clinical applications, because of countable limitations of existing synthetic drugs. Currently, flavonoids, glycoside and steroids like natural compounds have acquired great attention due to their anti-inflammatory and mucoregulatory effects. Most importantly, many natural compounds have shown their potential effects as the modulator of mucin expression, secretion, and production. Therefore, targeting airway MUC5AC expression and production represents an auspicious area of research for the development of drugs against various respiratory diseases.

Bronchial Epithelial Calcium Metabolism Impairment in Smokers and Chronic Obstructive Pulmonary Disease. Decreased ORAI3 Signaling.

The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke (CS), the major risk factor for developing chronic obstructive pulmonary disease (COPD). CS leads to bronchial epithelial damage on ciliated, goblet, and club cells, which could involve calcium (Ca2+) signaling. Ca2+ is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating, and epithelial repair. In this study, we analyzed Ca2+ signaling in air-liquid interface-reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired Ca2+ signaling. First, we showed that the endoplasmic reticulum (ER) depletion of Ca2+ stores was decreased in patients with COPD and that the Ca2+ influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute CS exposure led to a decrease in ER Ca2+ release, significant in smoker subjects, and to a decrease in Ca2+ influx only in control subjects. Furthermore, the differential expression of 55 genes involved in Ca2+ signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered Ca2+ influx and ciliary beating, but not mucus and cytokine secretion or epithelial repair, in control subjects. Our data suggest that Ca2+ signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.

TAS-203, an oral phosphodiesterase 4 inhibitor, exerts anti-inflammatory activities in a rat airway inflammation model.

Cyclic adenosine monophosphate (cAMP) is a key intracellular second messenger, which is degraded by phosphodiesterase 4 (PDE4). PDE4 suppresses cAMP levels, and thus stimulates the activity of inflammatory cells. Therefore, PDE4 has been considered as a therapeutic target for airway inflammatory diseases including asthma and chronic obstructive pulmonary disease (COPD). Roflumilast, an approved PDE4 inhibitor, has been shown to have clinical benefits in COPD. However, central nervous system-related side effects including nausea and vomiting have limited the therapeutic index of roflumilast. Moreover, although airway mucus hypersecretion is the characteristic feature, which is associated with the severity and prognosis, the inhibitory effect of roflumilast on sputum production is limited to a minority of patients. In this study, we demonstrate the inhibitory effects of TAS-203, which is an orally active PDE4 inhibitor associated with a lowered emetic effect, on airway inflammation and mucus hypersecretion. A cell-based assay showed TAS-203 treatment suppressed epidermal growth factor (EGF)-induced mucin MUC5AC expression. TAS-203 also suppressed monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-5 and IL-13 production in a Sephadex-induced airway inflammation model, and the number of infiltrating cells in bronchoalveolar lavage (BAL) fluid. TAS-203 caused marked reduction of goblet cell hyperplasia in a histopathological analysis of airway epithelium. Furthermore, TAS-203 suppressed 5-hydroxytryptamine (5-HT)-induced airway hyperresponsiveness (AHR). In addition, we preliminarily confirmed TAS-203 prevents airway MUC5AC production in BAL fluid, and shows lower specific airway resistance (sRaw) in a cigarette smoke-induced COPD-like model. Our data suggest that TAS-203 might be useful in the treatment of airway inflammatory disease.

Obtusifolin isolated from the seeds of Cassia obtusifolia regulates the gene expression and production of MUC5AC mucin in airway epithelial cells via affecting NF-κB pathway.

We investigated whether obtusin, obtusifolin, and cassiaside isolated from the seeds of Cassia obtusifolia inhibit the gene expression and production of airway mucin 5AC (MUC5AC). Confluent NCI-H292 cells were pretreated with obtusin, obtusifolin, or cassiaside for 30 min and then stimulated with epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA), or tumor necrosis factor-α (TNF-α) for 24 hr. The MUC5AC mucin gene expression was measured by reverse transcription-polymerase chain reaction. Production of MUC5AC mucin protein was measured by enzyme-linked immunosorbent assay. To elucidate the action mechanism of obtusifolin, effect of obtusifolin on PMA-induced nuclear factor kappa B (NF-κB) signaling pathway was investigated by western blot analysis. Obtusin, obtusifolin, or cassiaside inhibited the expression of MUC5AC mucin gene and the production of MUC5AC mucin protein, induced by EGF, PMA, or TNF-α. Obtusifolin inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase, and thus phosphorylation and degradation of inhibitory kappa B alpha. Obtusifolin inhibited PMA-induced nuclear translocation of NF-κB p65. These results suggest that obtusifolin can regulate the production and gene expression of mucin by acting on airway epithelial cells through regulation of NF-κB signaling pathway.

Expression of mucins (MUC1, MUC2, MUC5AC and MUC6) in ALK-positive lung cancer: Comparison with EGFR-mutated lung cancer.

ALK-positive (ALK+) lung adenocarcinoma usually shows a more advanced-staged disease with frequent nodal metastasis and highly aggressive outcomes compared with EGFR-mutated lung cancers. The aim of this study was to investigate the expression profiles of several mucins in ALK + lung cancers to gain insight into the relationship between the more aggressive biological nature of ALK + lung cancers and the role of mucins. We examined the immunohistochemical profiles of mucins MUC1, MUC2, MUC5AC, and MUC6 in 19 ALK + lung cancers compared with 42 EGFR-mutated lung cancers. ALK + cancers were found to occur in younger patients and were characterized by a solid-predominant histologic subtype with frequent signet ring cells and peritumoral muciphages. By contrast, EGFR-mutated cancers lacked ALK-specific histological patterns. Although all MUC1 and MUC5AC were expressed in both subtypes, MUC1 expression in ALK + cancers was visualized exclusively through cytoplasmic staining, whereas those in EGFR-mutated cancers were predominantly membranous staining in apical area (92.9%) and focally in cytoplasmic staining (7.1%). MUC5AC expression in ALK + cancers was exclusively visualized through cytoplasmic staining (100%), whereas EGFR-mutated cancers showed predominantly perinuclear dot-like patterns (90.5%) and focal cytoplasmic staining (9.5%). MUC2 and MUC6 expression was not detected in either type of lung cancer. CONCLUSIONS: The high frequency of both MUC1 and MUC5AC cytoplasmic expression, coupled with a lack of MUC2 and MUC6 expression in ALK + lung cancer may contribute to the biologically aggressive behavior of ALK + cancer. Inhibitors to these types of mucins may thus act as a barrier to cancerous extension reducing their aggressive behavior.

Effects of the Helicobacter pylori Virulence Factor CagA and Ammonium Ion on Mucins in AGS Cells.

PURPOSE: To investigate the effects of Helicobacter pylori (H. pylori)-CagA and the urease metabolite NH4⁺ on mucin expression in AGS cells. MATERIALS AND METHODS: AGS cells were transfected with CagA and/or treated with different concentrations of NH4CL. Mucin gene and protein expression was assessed by qPCR and immunofluorescence assays, respectively. RESULTS: CagA significantly upregulated MUC5AC, MUC2, and MUC5B expression in AGS cells, but did not affect E-cadherin and MUC6 expression. MUC5AC, MUC6, and MUC2 expression in AGS cells increased with increasing NH4⁺ concentrations until reaching a peak level at 15 mM. MUC5B mRNA expression in AGS cells (NH4⁺ concentration of 15 mM) was significantly higher than that at 0, 5, and 10 mM NH4⁺. No changes in E-cadherin expression in AGS cells treated with NH4⁺ were noted, except at 20 mM. The expression of MUC5AC, MUC2, and MUC6 mRNA in CagA-transfected AGS cells at an NH4⁺ concentration of 15 mM was significantly higher than that at 0 mM, and decreased at higher concentrations. The expression of MUC5B mRNA increased with increases in NH4⁺ concentration, and was significantly higher compared to that in untreated cells. No significant change in the expression of E-cadherin mRNA in CagA-transfected AGS cells was observed. Immunofluorescence assays confirmed the observed changes. CONCLUSION: H. pylori may affect the expression of MUC5AC, MUC2, MUC5B, and MUC6 in AGS cells via CagA and/or NH4⁺, but not E-cadherin.

Interleukin (IL) 36 gamma induces mucin 5AC, oligomeric mucus/gel-forming expression via IL-36 receptor-extracellular signal regulated kinase 1 and 2, and p38-nuclear factor kappa-light-chain-enhancer of activated B cells in human airway epithelial cells.

BACKGROUND: Mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) expression is significantly increased in allergic and inflammatory airway diseases. Interleukin (IL) 36 gamma is predominantly expressed in airway epithelial cells and plays an important role in innate and adaptive immune responses. IL-36 gamma is induced by many inflammatory mediators, including cytokines and bacterial and viral infections. However, the association between IL-36 gamma and mucin secretion in human airway epithelial cells has not yet been fully investigated. OBJECTIVE: The objective of this study was to determine whether IL-36 gamma might play a role in the regulation of mucin secretion in airway epithelial cells. We investigated the effect and brief signaling pathway of IL-36 gamma on MUC5AC expression in human airway epithelial cells. METHODS: Enzyme immunoassay, immunoblot analysis, immunofluorescence staining, reverse transcriptase-polymerase chain reaction (PCR), and real-time PCR were performed in mucin-producing human airway epithelial NCI-H292 cells and in human nasal epithelial cells after pretreatment with IL-36 gamma, several specific inhibitors, or small interfering RNAs (siRNA). RESULTS: IL-36 gamma induced MUC5AC expression and activated the phosphorylation of extracellular signal regulated kinase (ERK) 1 and 2, p38, and nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-kappa B). IL-36 receptor antagonist significantly attenuated these effects. The specific inhibitor and siRNA of ERK1, ERK2, p38, and NF-kappa B significantly attenuated IL-36 gamma induced MUC5AC expression. CONCLUSION: These results indicated that IL-36 gamma induced MUC5AC expression via the IL-36 receptor-mediated ERK1/2 and p38/NF-kappa B pathway in human airway epithelial cells.

Curcumin suppresses MUC5AC production via interfering with the EGFR signaling pathway.

Excessive mucin production in the airway may contribute to airway inflammatory diseases. Curcumin has been reported to prevent mucin 5AC (MUC5AC) production in human airway epithelial cells; however, the molecular targets of curcumin involved in regulating MUC5AC expression have remained elusive. The present study aimed to elucidate the molecular mechanisms by which curcumin regulates MUC5AC production, utilizing the NCI­H292 human airway epithelial cell line featuring MUC5AC hypersecretion. Curcumin was able to counteract the endothelial growth factor (EGF)­stimulated mRNA and protein expression of MUC5AC. In addition, curcumin treatment prevented EGF­induced phosphorylation of EGF receptor (EGFR) as well as the downstream AKT and signal transducer and activator of transcription 3 (STAT3), while inhibition of PI3K and STAT3 signaling significantly attenuated the expression of MUC5AC that was induced by EGF. Furthermore, EGF­induced increases in the levels of phosphorylated STAT3 in the nuclear fraction were inhibited by curcumin and PI3K inhibitors. In addition, treatment with curcumin significantly decreased MUC5AC and EGFR expression in a time­dependent manner under basal conditions. These results demonstrated that curcumin inhibited MUC5AC protein expression in NCI­H292 cells under basal conditions as well under EGF stimulation. This inhibition was accompanied by decreased activation of the EGFR/AKT/STAT3 pathway and reduced EGFR expression, which indicated that curcumin may have a dual role in interfering with the EGFR signaling pathway and inhibiting mucin expression in human airway epithelial cells.

The dopamine D1 receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium.

Background: Dopamine receptors comprise two subgroups, Gs protein-coupled "D1-like" receptors (D1, D5) and Gicoupled "D2-like" receptors (D2, D3, D4). In airways, both dopamine D1 and D2 receptors are expressed on airway smooth muscle and regulate airway smooth muscle force. However, functional expression of the dopamine D1 receptor has never been identified on airway epithelium. Activation of Gs-coupled receptors stimulate adenylyl cyclase leading to cyclic AMP (cAMP) production, which is known to induce mucus overproduction through the cAMP response element binding protein (CREB) in airway epithelial cells. We questioned whether the dopamine D1 receptor is expressed on airway epithelium, and whether it promotes CREB phosphorylation and MUC5AC expression. Methods: We evaluated the protein expression of the dopamine D1 receptor on native human airway epithelium and three sources of cultured human airway epithelial cells including primary cultured airway epithelial cells, the bronchial epithelial cell line (16HBE14o-), and the pulmonary mucoepidermoid carcinoma cell line (NCI-H292) using immunohistochemistry and immunoblotting. To characterize the stimulation of cAMP through the dopamine D1 receptor, 16HBE14o- cells and NCI-H292 cells were treated with dopamine or the dopamine D1 receptor agonists (SKF38393 or A68930) before cAMP measurements. The phosphorylation of CREB by A68930 in both 16HBE14o- and NCI-H292 cells was measured by immunoblot. The effect of dopamine or A68930 on the expression of MUC5AC mRNA and protein in NCI-H292 cells was evaluated by real-time PCR and immunofluorescence staining, respectively. Results: The dopamine D1 receptor protein was detected in native human airway epithelium and three sources of cultured human airway epithelial cells. Dopamine or the dopamine D1-like receptor agonists stimulated cAMP production in 16HBE14o- cells and NCI-H292 cells, which was reversed by the selective dopamine D1-like receptor antagonists (SCH23390 or SCH39166). A68930 significantly increased phosphorylation of CREB in both 16HBE14o- and NCI-H292 cells, which was attenuated by the inhibitors of PKA (H89) and MEK (U0126). Expression of MUC5AC mRNA and protein were also increased by either dopamine or A68930 in NCI-H292 cells. Conclusions: These results suggest that the activation of the dopamine D1 receptor on human airway epithelium could induce mucus overproduction, which could worsen airway obstructive symptoms.

Salvianolic acid B improves airway hyperresponsiveness by inhibiting MUC5AC overproduction associated with Erk1/2/P38 signaling.

Salvianolic acid B (SalB) is one of the main water-soluble composites from Chinese medicine Dansen (Radix miltiorrhiza). It is used for clinical treatment of various diseases including cardiovascular, lung, Liver, renal and cancers. However, the effects of SalB to allergy induced airway mucin hypersecretion, inflammation and hyperresponsiveness (AHR) remains not clear. Overproduction of airway MUC5AC is a central effector of inflammation that is strongly associated with AHR in asthmatic attack. In this study, we investigated the anti-asthmatic activity and mechanism of SalB in a murine model and human epithelial cells by monitoring changes in mucin expression and secretion, airway inflammation, AHR, and signaling pathways. SalB was administered by intragastric administration (i.g) daily for a week, starting at 21 days after sensitization of ovalbumin (OVA). All examinations were performed 24h after the last antigen challenge. We found that treatments with SalB significantly inhibited increase in the tracheobronchial secretion, glycosaminoglycan levels, interleukin (IL)-13, IL-4, and IL-5 cytokines mRNA and protein expression, and decrease in mucociliary clearance in lung tissues. Histological results demonstrated that SalB attenuated OVA-induced eosinophil infiltration, airway goblet cell hyperplasia, and MUC5AC and MUC5B mRNA and protein expression in lung tissues. SalB exhibited protective effects against AHR in OVA-challenged animals. In vitro, SalB significantly inhibited IL-13-induced MUC5AC and MUC5B mRNA and protein expression in human epithelial cells. These effects were blocked by SalB by downregulating the Erk1/2 and P38 signaling pathways. Taken together, these data indicate that treatment with SalB may improve AHR by inhibiting MUC5AC overproduction.

Compensatory increases in tear volume and mucin levels associated with meibomian gland dysfunction caused by stearoyl-CoA desaturase-1 deficiency.

The stearoyl-CoA desaturase (SCD) family of enzymes catalyzes monounsaturated fatty acid synthesis by inserting a cis double bond at the Δ9 position of saturated fatty acids. Disruption of these enzymes has been reported to induce a severe dry skin phenotype. Since lipid abnormalities in the meibomian glands have been associated with dry eye, we analyzed selected eye tissues contributing to tear volume and composition in genetically SCD-1-deficient mice (SCD-1 KO), including the lacrimal glands and conjunctiva. Previous histopathological analysis had revealed atrophy and loss of meibomian glands; taken together with the increased goblet cell and MUC5AC expression in the conjunctiva reported here, these findings suggest that the tear volume and mucin levels secreted are enhanced in the absence of lipid secretion as a compensatory mechanism. The expression of lipid metabolism genes in lacrimal glands was decreased in SCD1 KO mice. Thus, these results provide new pathophysiological mechanisms to pursue with regard to meibomian gland dysfunction. In addition, lack of SCD-1 causes a compensatory increase in the tear volume and mucin levels associated with changes in expression of lipid metabolism genes. These results may be useful as a new concept for dry eye treatment strategies.