Am J Respir Crit Care Med. 2018 Oct 23.
Okuda K, Chen G, Subramani DB, Wolf M, Gilmore RC, Kato T, Radicioni G, Kesimer M, Chua M, Dang H, Livraghi-Butrico A, Ehre C, Doerschuk CM, Randell SH, Matsui H, Nagase T, O'Neal WK, Boucher RC.
PMID: 30352166 | DOI: 10.1164/rccm.201804-0734OC
Abstract RATIONALE: MUC5AC and MUC5B are the predominant gel-forming mucins in the mucus layer of human airways. Each mucin has distinct functions and site-specific expression. However, the regional distribution of expression and cell types that secrete each mucin in normal/healthy human airways are not fully understood. OBJECTIVES: To characterize the regional distribution of MUC5B and MUC5AC in normal/healthy human airways and assess which cell types produce these mucins, referenced to the club cell secretory protein (CCSP). METHODS: Multiple airway regions from 16 non-smoker lungs without a history of lung disease were studied. MUC5AC, MUC5B, and CCSP expression/co-localization were assessed by RNA in situ hybridization (ISH) and immunohistochemistry in 5 lungs with histologically healthy airways. Droplet digital PCR and cell culture were performed for absolute quantification of MUC5AC/5B ratios and protein secretion, respectively. RESULTS: Submucosal glands expressed MUC5B, but not MUC5AC. However, MUC5B was also extensively expressed in superficial epithelia throughout the airways except for the terminal bronchioles. Morphometric calculations revealed that the distal airway superficial epithelium was the predominant site for MUC5B expression, whereas MUC5AC expression was concentrated in proximal, cartilaginous airways. RNA ISH revealed both MUC5AC and MUC5B were co-localized with CCSP-positive secretory cells in proximal superficial epithelia, whereas MUC5B and CCSP-co-positive cells dominated distal regions. CONCLUSIONS: In normal/healthy human airways, MUC5B is the dominant secretory mucin in the superficial epithelium as well as glands, with distal airways being a major site of expression. MUC5B and MUC5AC expression is a property of CCSP positive secretory cells in superficial airway epithelia.
Am J Respir Crit Care Med.
Chen G, Ribeiro CMP, Sun L, Okuda K, Kato T, Gilmore RC, Martino MB, Dang H, Abzhanova A, Lin JM, Hull-Ryde EA, Volmer AS, Randell SH, Livraghi-Butrico A, Deng Y, Scherer PE, Stripp BR, O'Neal WK, Boucher RC.
PMID: 30973754 | DOI: 10.1164/rccm.201810-1972OC
Abstract
RATIONALE:
The goal was to connect elements of IPF pathogenesis, including: chronic endoplasmic reticulum stress in respiratory epithelia associated with injury/inflammation and remodeling; distal airway mucus obstruction and honeycomb cyst formation with accumulation of MUC5B; and associations between IPF risk and polymorphisms in the MUC5B promoter.
OBJECTIVES:
Test whether the ER stress sensor protein ER-to-nucleus signaling 2 (ERN2) and its downstream effector, the spliced form of x-box binding protein 1 (XBP1S), regulate MUC5B expression and differentially activate the MUC5B promoter variant in respiratory epithelia.
METHODS AND MEASUREMENTS:
Primary human airway epithelia (HAE), transgenic mouse models, human IPF lung tissues, and cell lines expressing XBP1S and MUC5B promoters were used to explore relationships between the ERN2/XBP1S pathway and MUC5B. An inhibitor of the pathway, KIRA6, and XBP1 CRISPR-Cas9 were used in HAE to explore therapeutic potential.
RESULTS:
ERN2 regulated both MUC5B and MUC5AC mRNAs. Downstream XBP1S selectively promoted MUC5B expression in vitro and in distal murine airway epithelia in vivo. XBP1S bound to the proximal region of the MUC5B promoter and differentially up-regulated MUC5B expression in the context of the MUC5B promoter rs35705950 variant. High levels of ERN2 and XBP1S were associated with excessive MUC5B mRNAs in distal airways of human IPF lungs. Cytokine-induced MUC5B expression in HAE was inhibited by KIRA6 and XBP1 CRISPR-Cas9.
CONCLUSION:
A positive feedback bistable ERN2-XBP1S pathway regulates MUC5B-dominated mucus obstruction in IPF, providing a UPR-dependent mechanism linking the MUC5B promoter rs35705950 polymorphism with IPF pathogenesis. Inhibiting ERN2-dependent pathways/elements may provide a therapeutic option for IPF.
Devadoss, D;Acharya, A;Manevski, M;Houserova, D;Cioffi, M;Pandey, K;Nair, M;Chapagain, P;Mirsaeidi, M;Borchert, G;Byrareddy, S;Chand, H;
| DOI: 10.1016/j.isci.2022.104685
Noncoding RNAs are important regulators of mucoinflammatory response, but little is known about the contribution of airway long noncoding RNAs (lncRNAs) in COVID-19. RNA-seq analysis showed a more than four-fold increased expression of IL-6, ICAM-1, CXCL-8, and SCGB1A1 inflammatory factors; MUC5AC and MUC5B mucins; and SPDEF, FOXA3, and FOXJ1 transcription factors; in COVID-19 patient nasal samples compared to uninfected controls. A lncRNA on antisense strand to ICAM-1 or LASI was induced two-fold in COVID-19 patients and its expression was directly correlated with viral loads. A SARS-CoV-2 infected 3D-airway model largely recapitulated these clinical findings. RNA microscopy and molecular modeling indicated a possible interaction between viral RNA and LASI lncRNA. Notably, blocking LASI lncRNA reduced the SARS-CoV-2 replication and suppressed MUC5AC mucin levels and associated inflammation, and select LASI-dependent miRNAs (e.g., let-7b-5p and miR-200a-5p) were implicated. Thus, LASI lncRNA represents an essential facilitator of SARS-CoV-2 infection and associated airway mucoinflammatory response.
American journal of respiratory and critical care medicine
Kato, T;Asakura, T;Edwards, CE;Dang, H;Mikami, Y;Okuda, K;Chen, G;Sun, L;Gilmore, RC;Hawkins, P;De la Cruz, G;Cooley, MR;Bailey, AB;Hewitt, SM;Chertow, DS;Borczuk, AC;Salvatore, S;Martinez, FJ;Thorne, LB;Askin, FB;Ehre, C;Randell, SH;O'Neal, WK;Baric, RS;Boucher, RC;NIH COVID-19 Autopsy Consortium, ;
PMID: 35816430 | DOI: 10.1164/rccm.202111-2606OC
The incidence and sites of mucus accumulation, and molecular regulation of mucin gene expression, in COVID-19 lung disease have not been reported.Characterize incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease.Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by AB-PAS and immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. SARS-CoV-2-infected human bronchial epithelial (HBE) cultures were utilized to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures.MUC5B and variably MUC5AC RNA levels were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the sub-acute/chronic disease phase following SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of COVID-19 subjects 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 post inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days post inoculation. SARS-CoV-2 infection of HBE cultures induced expression of EGFR ligands and inflammatory cytokines (e.g., IL-1α/β) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways, or dexamethasone administration, reduced SARS-CoV-2-induced mucin expression.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 post 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. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Exp Mol Pathol. Feb 28. pii: S0014-4800(14)00023–9.
ørdal O, Qvigstad G, Nordrum IS, Sandvik AK, Gustafsson BI, Waldum H (2014)
PMID: 24589859 | DOI: 10.1016/j.yexmp.2014.02.008
PURPOSE:The purpose of this study is to assess the exocrine and neuroendocrine properties of tumour cells in diffuse gastric cancer with signet ring cell differentiation. MATERIAL AND METHODS: Mucin mRNA and protein expressions (MUC1, 2, 3, 4, 5AC, 6 and MUC13) were assessed by immunohistochemistry and in situ hybridization. The neuroendocrine properties were evaluated by protein and mRNA expression of the general neuroendocrine markers chromogranin A and synaptophysin. RESULTS: No MUC expression was observed in signet ring tumour cells including the amorphous substance in any of the nine cases. All cases showed immunoreactivity to synaptophysin, and seven out of nine cases immunoreactivity to chromogranin A in signet ring and non-signet ring tumour cells. Chromogranin A mRNA expression was observed in tumour cells in all samples with retained mRNA. CONCLUSIONS: The lack of MUC protein and mRNA in signet ring tumour cells suggests the amorphous substance is not mucin. The lack of MUC mRNA expression in non-signet ring tumour cells questions exocrine differentiation in this tumour group. The abundant protein expression of the general neuroendocrine markers CgA and synaptophysin, and mRNA expression in tumour cells strengthens the hypothesis that this tumour group may be of neuroendocrine origin.
Leushacke M, Tan SH, Wong A, Swathi Y, Hajamohideen A, Tan LT, Goh J, Wong E, Denil SLIJ, Murakami K, Barker N.
PMID: 28581476 | DOI: 10.1038/ncb3541
The daily renewal of the corpus epithelium is fuelled by adult stem cells residing within tubular glands, but the identity of these stem cells remains controversial. Lgr5 marks homeostatic stem cells and 'reserve' stem cells in multiple tissues. Here, we report Lgr5 expression in a subpopulation of chief cells in mouse and human corpus glands. Using a non-variegated Lgr5-2A-CreERT2 mouse model, we show by lineage tracing that Lgr5-expressing chief cells do not behave as corpus stem cells during homeostasis, but are recruited to function as stem cells to effect epithelial renewal following injury by activating Wnt signalling. Ablation of Lgr5+ cells severely impairs epithelial homeostasis in the corpus, indicating an essential role for these Lgr5+ cells in maintaining the homeostatic stem cell pool. We additionally define Lgr5+ chief cells as a major cell-of-origin of gastric cancer. These findings reveal clinically relevant insights into homeostasis, repair and cancer in the corpus.
Role for Mucin-5AC in Upper and Lower Airway Pathogenesis in Mice
Cho, HY;Park, S;Miller, L;Lee, HC;Langenbach, R;Kleeberger, SR;
PMID: 33938323 | DOI: 10.1177/01926233211004433
Mucin-5AC (MUC5AC) is a major secreted mucin in pathogenic airways. To determine its role in mucus-related airway disorders, Muc5ac-deficient (Muc5ac-/-) and wild-type (Muc5ac+/+) mice were compared in bleomycin-induced pulmonary fibrosis, respiratory syncytial virus (RSV) disease, and ozone toxicity. Significantly greater inflammation and fibrosis by bleomycin were developed in Muc5ac-/- lungs compared to Muc5ac+/+ lungs. More severe mucous cell metaplasia in fibrotic Muc5ac-/- lungs coincided with bronchial Muc2, Muc4, and Muc5b overexpression. Airway RSV replication was higher in Muc5ac-/- than in Muc5ac+/+ during early infection. RSV-caused pulmonary epithelial death, bronchial smooth muscle thickening, and syncytia formation were more severe in Muc5ac-/- compared to Muc5ac+/+. Nasal septal damage and subepithelial mucoserous gland enrichment by RSV were greater in Muc5ac-/- than in Muc5ac+/+. Ozone exposure developed more severe nasal airway injury accompanying submucosal gland hyperplasia and pulmonary proliferation in Muc5ac-/- than in Muc5ac+/+. Ozone caused periodic acid-Schiff-positive secretion only in Muc5ac-/- nasal airways. Lung E-cadherin level was relatively lower in Muc5ac-/- than in Muc5ac+/+ basally and after bleomycin, RSV, and ozone exposure. Results indicate that MUC5AC is an essential mucosal component in acute phase airway injury protection. Subepithelial gland hyperplasia and adaptive increase of other epithelial mucins may compensate airway defense in Muc5ac-/- mice.
Sato, Y;Ban, S;Katayama, Y;Mitsui, T;
PMID: 35430166 | DOI: 10.1016/j.humpath.2022.04.001
The aim of this study was to clarify the correlation between gastrin receptor (GR) expression in the gastric oxyntic mucosa and fundic gland polyps (FGPs) and the histological and immunohistochemical findings of the mucosa as well as the history of proton pump inhibitor (PPI) administration. The unique membranous linear positivity of GR in parietal cells was reproducibly observed by immunohistochemistry, which was also validated by immunofluorescence. Further histological and immunohistochemical examination of 34 oxyntic mucosae and 43 FGPs revealed the following: 1) parietal cells (PCs) with membranous linear GR expression (mGR) was observed to be limited to the isthmus-neck region in the normal state; 2) Appearance of PCs with mGR in the deep oxyntic gland regions was significantly related to the PPI medication history; 3) PCs with mGR were more frequently observed in the deep oxyntic gland regions when the oxyntic mucosa showed derangement of mucosal component cell compartmentalization revealed by MUC5AC and MUC6 immunohistochemistry, which was also significantly related to the PPI use; and 4) PCs with intense membranous linear positivity of GR were observed to be diffusely distributed in all of the cases of FGPs. In conclusion, the distribution of unique GR membranous linear expression in PCs of the oxyntic mucosa under PPI medication and FGPs could reflect the pathologic mucosal state characterized by derangement of the compartmentalization of mucosal component cells, which could be another basis for evaluating physiologic and/or pathophysiologic conditions of the gastric mucosa.
Alteration of Colonic Mucin Composition and Cytokine Expression in Acute Swine Dysentery
Lin, SJ;Arruda, B;Burrough, E;
PMID: 33686884 | DOI: 10.1177/0300985821996657
Swine dysentery (SD) is an enteric disease associated with strongly β-hemolytic Brachyspira spp. that cause mucohemorrhagic diarrhea primarily in grower-finisher pigs. We characterized alteration of colonic mucin composition and local cytokine expression in the colon of pigs with acute SD after B. hyodysenteriae (Bhyo) infection and fed either a diet containing 30% distillers dried grains with solubles (DDGS) or a control diet. Colonic tissue samples from 9 noninoculated pigs (Control, N = 4; DDGS, N = 5) and 10 inoculated pigs experiencing acute SD (Bhyo, N = 4; Bhyo-DDGS, N = 6) were evaluated. At the apex of the spiral colon, histochemical staining with high-iron diamine-Alcian blue revealed increased sialomucin (P = .008) and decreased sulfomucin (P = .027) in Bhyo pigs relative to controls, with a dietary effect for sulfomucin. Noninoculated pigs fed DDGS had greater expression of sulfomucin (P = .002) compared to pigs fed the control diet. Immunohistochemically, there was de novo expression of mucin 5AC (MUC5AC) in the Bhyo group while mucin 2 (MUC2) expression was not significantly different between groups. RNA in situ hybridization to detect the pro-inflammatory cytokine IL-1β often showed increased expression in the Bhyo group although without statistical significance, and this was not correlated with MUC5AC or MUC2 expression, suggesting IL-1β is not a major regulator of their secretion in acute SD. Expression of the anti-inflammatory cytokine TGF-β1 was significantly suppressed in the Bhyo group compared to controls (P = .005). This study reveals mucin and cytokine alterations in the colon of pigs with experimentally induced SD and related dietary effects of DDGS.
