ATF4-mediated GDF15 suppresses LPS-induced inflammation and MUC5AC in human nasal epithelial cells through the PI3K/Akt pathway.

AIMS: Growth and differentiation factor 15 (GDF15) is a stress-related factor, which implicated in various diseases. This study aimed to investigate the role of GDF15 in LPS-mediated inflammation and to explore the potential underlying molecular mechanisms in human nasal epithelial cells (HNEpCs). MAIN METHODS: HNEpCs were treated with LPS. GDF15 loss-of-function and gain-of-function experiments were performed. The expression of GDF15 by quantitative real-time PCR (RT-qPCR). The mRNA levels and secretion of inflammatory cytokines and MUC5AC were assessed by RT-qPCR and ELISA kits. LY294002 (PI3K inhibitor) and 740Y-P (PI3K agonist) were utilized to interfere with PI3k/Akt pathway. The relationship between GDF15 and ATF4 was identified by chromatin immunoprecipitation (ChIP) and luciferase reporter assay. KEY FINDINGS: We observed that LPS triggered GDF15 expression. GDF15 ablation reduced the mRNA levels and secretion of inflammatory cytokines. GDF15 silencing led to the reduction of the MUC5AC mRNA level, protein level and secretion in response to LPS. Enhanced expression of GDF15 showed the opposite results. Furthermore, we found that GDF15 deficiency inhibited activation of the PI3K/Akt pathway, LY294002 treatment further enhanced the role of GDF15 suppression in inflammation and MUC5AC expression, while 740Y-P administration partly reversed the biological activities of GDF15 silencing. ATF4 could bind to the promoter of GDF15 and positively regulate GDF15 expression. Depression of ATF4 diminished the secretion of inflammatory cytokines and MUC5AC via regulation of GDF15. SIGNIFICANCE: Our data suggest that GDF15 is regulated by ATF4 and suppresses LPS-induced inflammation and MUC5AC in human nasal epithelial cells through the PI3K/Akt pathway.

Secretory Mucin 5AC Promotes Neoplastic Progression by Augmenting KLF4-Mediated Pancreatic Cancer Cell Stemness.

Secreted mucin 5AC (MUC5AC) is the most abundantly overexpressed member of the mucin family during early pancreatic intraepithelial neoplasia stage I (PanIN-I) of pancreatic cancer. To comprehend the contribution of Muc5ac in pancreatic cancer pathology, we genetically ablated it in an autochthonous murine model (KrasG12D; Pdx-1cre, KC), which mirrors the early stages of pancreatic cancer development. Neoplastic onset and the PanIN lesion progression were significantly delayed in Muc5ac knockout (KrasG12D; Pdx-1 cre; Muc5ac-/-, KCM) animals with a 50% reduction in PanIN-2 and 70% reduction in PanIN-3 lesions compared with KC at 50 weeks of age. High-throughput RNA-sequencing analysis from pancreatic tissues of KCM animals revealed a significant decrease in cancer stem cell (CSC) markers Aldh1a1, Klf4, EpCAM, and CD133. Furthermore, the silencing of MUC5AC in human pancreatic cancer cells reduced their tumorigenic propensity, as indicated by a significant decline in tumor formation frequency by limiting dilution assay upon subcutaneous administration. The contribution of MUC5AC in CSC maintenance was corroborated by a significant decrease in tumor burden upon orthotopic implantation of MUC5AC-depleted pancreatic cancer cells. Mechanistically, MUC5AC potentiated oncogenic signaling through integrin αvß5, pSrc (Y416), and pSTAT3 (Y705). Phosphorylated STAT3, in turn, upregulated Klf4 expression, thereby enriching the self-renewing CSC population. A strong positive correlation of Muc5ac with Klf4 and pSTAT3 in the PanIN lesions of KC mouse pancreas reinforces the crucial involvement of MUC5AC in bolstering the CSC-associated tumorigenic properties of Kras-induced metaplastic cells, which leads to pancreatic cancer onset and progression. SIGNIFICANCE: This study elucidates that de novo expression of MUC5AC promotes cancer cell stemness during Kras-driven pancreatic tumorigenesis and can be targeted for development of a novel therapeutic regimen.

Muc5ac null mice are predisposed to spontaneous gastric antro-pyloric hyperplasia and adenomas coupled with attenuated H. pylori-induced corpus mucous metaplasia.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide and is strongly associated with chronic Helicobacter pylori (Hp) infection. The ability of Hp to closely adhere to the gastric surface protective mucous layer containing mucins (MUC in humans and Muc in animals), primarily Muc5ac, is integral in the stepwise pathogenesis from gastritis to cancer. To probe the role of Muc5ac in Hp-induced gastric pathology, Muc5ac-/- and Muc5ac+/+ (WT) mice were experimentally infected with Hp Sydney strain (SS1). At 16 weeks and 32 weeks post infection (wpi), groups of mice were euthanized and evaluated for the following: gastric histopathological parameters, immunohistochemical expression of mucins (Muc5ac, Muc1, Muc2), Trefoil factor family proteins (Tff1 and Tff2), Griffonia (Bandeiraea) simplicifolia lectin II (GSL II) (mucous metaplasia marker) and Clusterin (Spasmolytic Polypeptide Expressing Metaplasia (SPEM) marker), Hp colonization density by qPCR and gastric cytokine mRNA levels. Our results demonstrate that Muc5ac-/- mice developed spontaneous antro-pyloric proliferation, adenomas and in one case with neuroendocrine differentiation; these findings were independent of Hp infection along with strong expression levels of Tff1, Tff2 and Muc1. Hp-infected Muc5ac-/- mice had significantly lowered gastric corpus mucous metaplasia at 16 wpi and 32 wpi (P = 0.0057 and P = 0.0016, respectively), with a slight reduction in overall gastric corpus pathology. GSII-positive mucous neck cells were decreased in Hp-infected Muc5ac-/- mice compared to WT mice and clusterin positivity was noted within metaplastic glands in both genotypes following Hp infection. Additionally, Hp colonization densities were significantly higher in Muc5ac-/- mice compared to WT at 16 wpi in both sexes (P = 0.05) along with a significant reduction in gastric Tnfα (16 wpi-males and females, P = 0.017 and P = 0.036, respectively and 32 wpi-males only, P = 0.025) and Il-17a (16 wpi-males) (P = 0.025). Taken together, our findings suggest a protective role for MUC5AC/Muc5ac in maintaining gastric antral equilibrium and inhibiting Hp colonization and associated inflammatory pathology.

Control of lung defence by mucins and macrophages: ancient defence mechanisms with modern functions.

Owing to the need to balance the requirement for efficient respiration in the face of tremendous levels of exposure to endogenous and environmental challenges, it is crucial for the lungs to maintain a sustainable defence that minimises damage caused by this exposure and the detrimental effects of inflammation to delicate gas exchange surfaces. Accordingly, epithelial and macrophage defences constitute essential first and second lines of protection that prevent the accumulation of potentially harmful agents in the lungs, and under homeostatic conditions do so effectively without inducing inflammation. Though epithelial and macrophage-mediated defences are seemingly distinct, recent data show that they are linked through their shared reliance on airway mucins, in particular the polymeric mucin MUC5B. This review highlights our understanding of novel mechanisms that link mucus and macrophage defences. We discuss the roles of phagocytosis and the effects of factors contained within mucus on phagocytosis, as well as newly identified roles for mucin glycoproteins in the direct regulation of leukocyte functions. The emergence of this nascent field of glycoimmunobiology sets forth a new paradigm for considering how homeostasis is maintained under healthy conditions and how it is restored in disease.

MUC5AC interactions with integrin ß4 enhances the migration of lung cancer cells through FAK signaling.

MUC5AC is a secretory mucin aberrantly expressed in various cancers. In lung cancer, MUC5AC is overexpressed in both primary and metastatic lesions; however, its functional role is not well understood. The present study was aimed at evaluating mechanistic role of MUC5AC on metastasis of lung cancer cells. Clinically, the overexpression of MUC5AC was observed in lung cancer patient tissues and was associated with poor survival. In addition, the overexpression of Muc5ac was also observed in genetically engineered mouse lung adenocarcinoma tissues (Kras(G12D); Trp53(R172H/+); AdCre) in comparison with normal lung tissues. Our functional studies showed that MUC5AC knockdown resulted in significantly decreased migration in two lung cancer cell lines (A549 and H1437) as compared with scramble cells. Expression of integrins (α5, ß1, ß3, ß4 and ß5) was decreased in MUC5AC knockdown cells. As both integrins and MUC5AC have a von Willebrand factor domain, we assessed for possible interaction of MUC5AC and integrins in lung cancer cells. MUC5AC strongly interacted only with integrin ß4. The co-localization of MUC5AC and integrin ß4 was observed both in A549 lung cancer cells as well as genetically engineered mouse adenocarcinoma tissues. Activated integrins recruit focal adhesion kinase (FAK) that mediates metastatic downstream signaling pathways. Phosphorylation of FAK (Y397) was decreased in MUC5AC knockdown cells. MUC5AC/integrin ß4/FAK-mediated lung cancer cell migration was confirmed through experiments utilizing a phosphorylation (Y397)-specific FAK inhibitor. In conclusion, overexpression of MUC5AC is a poor prognostic marker in lung cancer. MUC5AC interacts with integrin ß4 that mediates phosphorylation of FAK at Y397 leading to lung cancer cell migration.

[Effect of methyleugenol on expression of MUC5AC in nasal mucosa of rats with allergic rhinitis].

Objective: To investigate the effect of methyleugenol on expression of MUC5AC in nasal mucosa of rats with allergic rhinitis (AR). Methods: Seventy-two Wistar rats were randomly divided into 6 groups:normal control group, AR group, loratadine group, low-dose methyleugenol group, middle-dose methyleugenol group and high-dose methyleugenol group with 12 rats in each group. AR was induced by intraperitoneal injection of ovalbumin in latter 5 groups. 10 mg loratadine q.d was given to rats in loratadine group by gavage; and 10 mg/kg, 20 mg/kg and 40 mg/kg methyleugenol were given by gavege q.d to rats in low-, middle-and high-dose methyleugenol groups, respectively. Nasal mucosa samples were obtained from rats at 1, 2, 4 and 6 weeks after drug intervention. The expression of MUC5AC protein and mRNA in nasal mucosa was detected by immunohistochemistry and real-time fluorescence quota PCR (RT-PCR), respectively. Results: Compared with AR, the percentage of cells staining positively for MUC5AC protein and the relative quantity of MUC5AC mRNA in middle-and high-dose methyleugenol groups were significantly decreased after 2 and 4 weeks of drug intervention (P<0.05), but no such decrease was observed in low-dose methyleugenol group at all time points (P>0.05). The percentage of cells with positive expression of MUC5AC protein and mRNA in loratadine group were significantly decreased after 1 week of administration (P<0.05). The percentage of cells with positive MUC5AC protein in middle-dose methyleugenol group was higher than that in loratadine group (P<0.05) after 6 week of drug intervention, but the difference was not seen in high-dose group (P>0.05). There was no significant difference in relative quantities of MUC5AC mRNA after 4 weeks of administration between high-and middle-dose methyeugenol groups and loratadine group (P>0.05). Conclusion: Methyleugenol can attenuate AR through inhibiting the expression of MUC5AC mRNA and protein in nasal mucosa of AR rats.

[Influence of hot-humid stress and acclimation on airway mucus production in lungs of mice].

OBJECTIVE: To study the change of airway mucus secretion under a high temperature and humidity environment, and explore the effects of hot-humid stress and acclimation on the morbidity and mortality of respiratory disease. METHODS: Forty-five BABL/c mice were randomly divided into five groups:normal group, hot-humid group I, hot-humid group Ⅱ, hot-humid group Ⅲ, hot-humid group IV, with 9 mice in each group. Mice in normal group were continuously placed in the common environment and sacrificed after 7 days. Mice in other groups were housed in a temperature-and-humidity-controlled environment (33℃±0.5℃, 95%±5%). Mice in hot-humid group I, hot-humid group Ⅱ, hot-humid group Ⅲ and hot-humid group IV were sacrificed after 12 hours, 24 hours, 4 days, 7 days respectively. The protein expression of mucin 5AC(MUC5AC)、epidermal growth factor receptor (EGFR)、aquaporin 1(AQP1) and aquaporin 5(AQP5) in lung were tested by immunohistochemisty. RESULTS: After housed in a high temperature and humidity environment, immunohistochemisty revealed a significant increase of AQP5 12 h later, MUC5AC and EGFR 24 h later, compared with normal group(P<0.05). There was a significant decrease of MUC5AC 7 d later, compared with normal group(P<0.05). There was no significant difference in MUC5AC, EGFR and AQP5 expression among all groups at other time points. There was no difference of AQP1 in humid heat groups, compared with normal group, but a significant decrease in humid heat Ⅲ and IV groups, compared with humid heat I and Ⅱ groups. CONCLUSIONS: These findings indicate that hot-humid stress induces mucus hypersecretion in airways, which may be related to the up-regulation of EGFR and down-regulation of AQP5 in MUC5AC. Although acclimation mitigates above-mentioned response, a series of more complex responses may be induced if still in the hot-humid environment.

Effect of glycosides based standardized fenugreek seed extract in bleomycin-induced pulmonary fibrosis in rats: Decisive role of Bax, Nrf2, NF-κB, Muc5ac, TNF-α and IL-1ß.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive multifactorial disease with limited therapeutic options. Glycosides based standardized fenugreek seed extract (SFSE-G) possesses potent anti-inflammatory and anti-oxidant property. AIM: To evaluate the efficacy of SFSE-G against bleomycin (BLM) induced pulmonary fibrosis by assessing behavioral, biochemical, molecular and ultrastructural changes in the laboratory rats. MATERIALS AND METHODS: IPF was induced in male Sprague-Dawley rats by single intratracheal BLM (6IU/kg) injection followed by SFSE-G (5, 10, 20 and 40mg/kg, p.o.) or methylprednisolone (10mg/kg, p.o.) treatment for 28day. Various parameters were analyzed in lung and bronchoalveolar lavage fluid (BALF) after 14 and 28days of the drug treatment. RESULTS: SFSE-G (20 and 40mg/kg, p.o.) administration significantly prevented the BLM induced alteration in body weight, lung index, lung function test and hematology. The altered total and differential cell count in BALF and blood was significantly prevented by SFSE-G treatment. The decreased peripheral blood oxygen content after BLM instillation was significantly increased by SFSE-G treatment. SFSE-G significantly enhanced the BALF and lung antioxidant status, through modulating the SOD, GSH, T-AOC, MDA, NO level and Nrf2, HO-1 mRNA expression. There was a significant reduction in lung 5-HT level by SFSE-G treatment. The altered mRNA expression of biomarkers of lung inflammation (TNF-α, IL-1ß, IL-6 and IL-8), fibrosis (TGF-ß, collagen-1, ET-1, Muc5ac, NF-κB, VEGF, Smad-3) and apoptosis (Bax, Bcl-2 and Caspase-3) were significantly prevented by SFSE-G treatment. BLM induced histological inflammatory and fibrotic insult in the lung were reduced by SFSE-G treatment. It also ameliorated BLM induced lung ultrastructural changes as observed by transmission electron microscopic studies. However, administration of SFSE-G (5mg/kg, p.o.) failed to show any protective effect against BLM-induced PF whereas SFSE-G (10mg/kg, p.o.) showed significant amelioration in BLM-induced PF except lung function test, BALF and lung antioxidant level. CONCLUSION: SFSE-G showed anti-fibrotic efficacy executed through induction of Nrf2, which in turn may modulate anti-inflammatory molecules, inhibit fibrogenic molecules and decreased apoptosis to ameliorate BLM induced pulmonary fibrosis.

[Expression and role of sugar chains on airway mucus during the exacerbation of airway inflammation].

Human bronchial mucins, such as MUC5AC, have traditionally been defined as a family of high-molecular weight glycoproteins. Changes in the contents of sugar chains on MUC5AC are among the fundamental features in inflammatory respiratory disease. The changes have been shown to lead to unfavorable alterations in the viscosity of mucus, resulting in impairment of mucociliary transport, vulnerability to viral/bacterial infection as sugar chains play an important role in adhesion of some viruses and bacteria to the epithelium, and finally inflammatory cell infiltration in the airway. Recently, we found that expression of some glycosyltransferases associated with the contents and structure of sugar chains is regulated by phosphatidylinositol-phospholipase (PI-PL) C signaling in cells. L-Carbocisteine, a mucoregulatory drug, normalized or balanced fucosylated and sialylated sugar chains, such as sialyl Lewis x through inhibition of PI-PL C signaling. We prepared MUC5AC fusion protein with tandem repeats associated with MUC5AC, and confirmed that L-carbocisteine inhibited the increases in viscosity associated with sialyl Lewis x expression levels. In addition, the clinical study (2008) noted that L-carbocisteine reduced the frequency of common colds and exacerbation of symptoms in patients with COPD. These favorable effects in patients may be due to normalization of sugar chain contents on mucins. We suggest that the inhibitory effect on infection of airway epithelial cells by rhinoviruses, respiratory syncytial virus, and influenza viruses by treatment with L-carbocisteine may also be based on the regulation of sugar chain contents or structures on mucins.

The relationship among PDX1, CDX2, and mucin profiles in gastric carcinomas; correlations with clinicopathologic parameters.

PURPOSE: Several studies performed on pancreatic-duodenal homeobox 1 (PDX1) have demonstrated a loss of expression and negative tumor modulator effect in gastric carcinoma. Relations between PDX1 and gastric metaplasia, differentiated type of gastric carcinoma, and the early stage of the disease have been exhibited in previous reports. The aim of this study was to examine expressions of PDX1, caudal type homeobox 2 (CDX2) and mucin (MUC) profiles to address the role of PDX1 in gastric carcinogenesis and its relationship with CDX2. METHODS: Seventy gastrectomy specimens were analyzed immunohistochemically for PDX1, CDX2, MUC2, MUC5AC, and MUC6 expressions. The sum of cytoplasmic and nuclear PDX1 immunostaining and PDX1 positivity were assessed. All of the antibodies were examined for a correlation with tumor type, clinicopathologic parameters, and metaplasias. The relation of Ki-67 proliferation index with the expression profiles was also investigated. RESULTS: Neither PDX1 (66/70) nor CDX2 (37/70) and the mucin profiles (MUC2:11/70, MUC5AC:48/70, MUC6:41/70) showed a significant difference between differentiated and undifferentiated types of gastric carcinoma and clinicopathologic parameters. The PDX1 expression frequency was 94.3%, with an average PDX1 score of 8.8 ± 4.2. PDX1 and CDX2 expression showed a significant difference (P = 0.026 and P = 0.002, respectively) among the phenotypic classification of gastric carcinomas. All of the gastric and intestinal mixed-phenotype gastric carcinomas (GI-type) showed both PDX1 and CDX2 immunopositivity. Except for the relation of PDX1 score with MUC6 expression, no significant difference was detected between PDX1 and CDX2, MUC2, and MUC5AC expressions. A relationship between CDX2 and MUC2 and also between MUC5AC and MUC6 was found statistically. The Ki-67 proliferation index revealed a significant positive correlation with PDX1, CDX2, and MUC2 positivity. CONCLUSIONS: PDX1 expression revealed a higher positivity in gastric carcinomas than the previous studies and showed no relation with tumor type, clinicopathologic parameters, CDX2 expression, or mucin profiles. However, a significant relation of PDX1 and CDX2 expressions among phenotypic classification of gastric carcinomas reveals an idea about similar functions for PDX1 and CDX2 in the evolution of gastric carcinoma.

Tumor-associated MUC5AC stimulates in vivo tumorigenicity of human pancreatic cancer.

MUC5AC, a high molecular weight glycoprotein, is overexpressed in the ductal region of human pancreatic cancer but is not detectable in the normal pancreas, suggesting its association with disease development. In the present study, we investigated the in vitro and in vivo effects of MUC5AC knockdown by short interfering RNA (siRNA) in the MUC5AC-overexpressing SW1990 and BxPC3 human pancreatic cancer cell lines in order to clarify its function. Significant decreases in the expression levels of MUC5AC mRNA and protein were observed in SW1990 and BxPC3 cells that had been stably transfected with a MUC5AC siRNA expression vector (SW1990/si-MUC5AC and BxPC3/si-MUC5AC cells) compared to those in cells transfected with an si-mock vector (SW1990/si-mock and BxPC3/si-mock cells). In in vitro studies, neither type of MUC5AC-knockdown cell showed any difference in cell survival, proliferation, or morphology from the si-mock cells or parental cells. However, in vivo xenograft studies demonstrated that MUC5AC knockdown significantly reduced the tumorigenicity and suppressed the tumor growth of si-MUC5AC cells compared to those of the si-mock cells. Immunohistochemical analysis revealed that CD45R/B220+ and Gr-1+ cells had infiltrated into the tumor tissue of the SW1990/si-MUC5AC cells. Furthermore, cancer-associated antigen specific antibodies were detected at high levels in the sera from the SW1990/si-MUC5AC cell-bearing mice. These results suggest that tumor-associated MUC5AC expressed on the surface of pancreatic cancer cells supports the escape of pancreatic cancer cells from immunosurveillance. The present findings highlight a new dimension of MUC5AC as a functional immunosuppressive agent and its important role in pancreatic cancer progression.

Role of mucins in the skin during benign and malignant conditions.

Skin-related diseases comprise a major health challenge to the practicing physician, and constitute a significant psychological, social and financial burden to the society. Further, skin cancer, especially non-melanoma skin cancer is currently the leading type of malignancy in the Western world. Given the huge burden of skin diseases, there is growing emphasis on understanding their pathophysiology, and towards their early detection. Mucins are high-molecular weight O- and N-linked glycoproteins that have emerged in recent years as important molecules in maintaining health and in promoting or protecting against inflammation and cancer. They have also begun to emerge as highly specific diagnostic and prognostic markers and novel therapeutic targets in several malignant disorders. However, their role in cutaneous pathologies has remained largely obscured. The present review provides the expression patterns and proposed role of mucins in the healthy skin and various benign and malignant skin diseases. The review has immense clinical significance as the availability of highly specific reagents including monoclonal antibodies against mucins makes them extremely attractive targets for specific diagnosis and/or immunotherapy of benign and malignant cutaneous diseases.