Resolvin D2 and Resolvin D1 Differentially Activate Protein Kinases to Counter-Regulate Histamine-Induced [Ca2+]i Increase and Mucin Secretion in Conjunctival Goblet Cells

Resolvin (Rv) D2 and RvD1 are biosynthesized from docosahexaenoic acid (DHA) and promote resolution of inflammation in multiple organs and tissues, including the conjunctiva. Histamine is a mediator produced by mast cells in the conjunctiva during the allergic response. We determined the interaction of RvD2 with histamine and its receptor subtypes in cultured conjunctival goblet cells and compared them with RvD1 by measuring intracellular [Ca2+] and mucous secretion. Treatment with RvD2 significantly blocked the histamine-induced [Ca2+]i increase as well as secretion. RvD2 and RvD1 counter-regulate different histamine receptor subtypes. RvD2 inhibited the increase in [Ca2+]i induced by the activation of H1, H3, or H4 receptors, whereas RvD1 inhibited H1 and H3 receptors. RvD2 and RvD1 also activate distinct receptor-specific protein kinases to counter-regulate the histamine receptors, probably by phosphorylation. Thus, our data suggest that the counter-regulation of H receptor subtypes by RvD2 and RvD1 to inhibit mucin secretion are separately regulated.

Laxative Effects of Phlorotannins Derived from Ecklonia cava on Loperamide-Induced Constipation in SD Rats

This study investigated the laxative effects of phlorotannins (Pt) derived from Ecklonia cava (E. cave) on chronic constipation by evaluating alterations in stool parameters, gastrointestinal motility, histopathological structure, mucin secretion, gastrointestinal hormones, muscarinic cholinergic regulation, and fecal microbiota in SD rats with loperamide (Lop)-induced constipation subjected to Pt treatment. Stool-related parameters (including stool number, weight, and water contents), gastrointestinal motility, and length of intestine were significantly enhanced in the Lop+Pt-treated group as compared to the Lop+Vehicle-treated group. A similar recovery was detected in the histopathological and cytological structure of the mid-colon of Lop+Pt-treated rats, although the level of mucin secretion remained constant. Moreover, rats with Lop-induced constipation subjected to Pt treatment showed significant improvements in water channel expression, gastrointestinal hormone secretions, and expression of muscarinic acetylcholine receptors M2/M3 (mAChRs M2/M3) and their mediators of muscarinic cholinergic regulation. Furthermore, the Lop+Pt-treated group showed a significant recovery of Bifidobacteriaceae, Muribaculaceae, Clostridiaceae, and Eubacteriaceae families in fecal microbiota. Taken together, these results provide the first evidence that exposure of SD rats with Lop-induced constipation to Pt improves the constipation phenotype through the regulation of membrane water channel expression, GI hormones, the mAChR signaling pathway, and fecal microbiota.

Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis

Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh) dependent endocytic event remarkable for delivery of fluid phase cargo retrograde into the trans golgi network and across the cell by transcytosis - in addition to the expected transport of fluid phase cargo by endosomes to multi-vesicular bodies and lysosomes. While ACh also induced goblet cells to secrete mucins, ACh-induced GAP formation and mucin secretion were functionally independent and mediated by different receptors and signaling pathways, enabling goblet cells to differentially regulate these processes to accommodate the dynamically changing demands of the mucosal environment for barrier maintenance and sampling of lumenal substances.

Reversing chemorefraction in colorectal cancer cells by controlling mucin secretion

ABSTRACT15% of colorectal cancers (CRC) cells exhibit a mucin hypersecretory phenotype, which is suggested to provide resistance to immune surveillance and chemotherapy. We now formally show that colorectal cancer cells build a barrier to chemotherapeutics by increasing mucins’ secretion. We show that low levels of KChIP3, a negative regulator of mucin secretion (Cantero-Recasens et al., 2018), is a risk factor for CRC patients’ relapse in subset of untreated tumours. Our results also reveal that cells depleted of KChIP3 are four times more resistant (measured as cell viability and DNA damage) to chemotherapeutics 5-Fluorouracil plus Irinotecan (5-FU+iri.) compared to control cells, whereas KChIP3 overexpressing cells are 10 times more sensitive to killing by chemotherapeutics. Similar increase in tumour cell death is observed upon chemical inhibition of mucin secretion by the sodium/calcium exchanger (NCX) blockers (Mitrovic et al., 2013). Finally, sensitivity of CRC patient-derived organoids to 5-FU+iri increases 40-fold upon mucin secretion inhibition. Reducing mucin secretion thus provides a means to control chemoresistance of mucinous colorectal cancer cells and other mucinous tumours.

Bordetella Adenylate Cyclase Toxin Elicits Airway Mucin Secretion through Activation of the cAMP Response Element Binding Protein

The mucus layer protects airway epithelia from damage by noxious agents. Intriguingly, Bordetella pertussis bacteria provoke massive mucus production by nasopharyngeal epithelia during the initial coryza-like catarrhal stage of human pertussis and the pathogen transmits in mucus-containing aerosol droplets expelled by sneezing and post-nasal drip-triggered cough. We investigated the role of the cAMP-elevating adenylate cyclase (CyaA) and pertussis (PT) toxins in the upregulation of mucin production in B. pertussis-infected airway epithelia. Using human pseudostratified airway epithelial cell layers cultured at air–liquid interface (ALI), we show that purified CyaA and PT toxins (100 ng/mL) can trigger production of the major airway mucins Muc5AC and Muc5B. Upregulation of mucin secretion involved activation of the cAMP response element binding protein (CREB) and was blocked by the 666-15-Calbiochem inhibitor of CREB-mediated gene transcription. Intriguingly, a B. pertussis mutant strain secreting only active PT and producing the enzymatically inactive CyaA-AC– toxoid failed to trigger any important mucus production in infected epithelial cell layers in vitro or in vivo in the tracheal epithelia of intranasally infected mice. In contrast, the PT– toxoid-producing B. pertussis mutant secreting the active CyaA toxin elicited a comparable mucin production as infection of epithelial cell layers or tracheal epithelia of infected mice by the wild-type B. pertussis secreting both PT and CyaA toxins. Hence, the cAMP-elevating activity of B. pertussis-secreted CyaA was alone sufficient for activation of mucin production through a CREB-dependent mechanism in B. pertussis-infected airway epithelia in vivo.

Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca2+ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases

The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion and to identify the signaling pathways. The increase in [Ca2+]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca2+]i and mucin secretion that was blocked by the cell-permeant Ca2+ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca2+]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca2+]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca2+/ATPase that causes the intracellular Ca2+ stores to empty, and blockade of Ca2+ influx. Inhibition of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca2+]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca2+]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca2+]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca2+]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.

Oral administration of polystyrene microplastics for 2 weeks induces chronic constipation in ICR mice

Objective: Indirect evidence has determined the possibility that microplastics (MP) induce constipation, although direct scientific proof for constipation induction in animals remains unclear. Thus, this study is aimed to investigate whether oral administration of polystyrene MP causes constipation.Methods: An alteration in the constipation parameters and their molecular mechanisms was analyzed in ICR mice treated with 0.5 μm polystyrene (PS)-MP for 2 weeks. Results: Significant alterations in water consumption, stool weight, stool water contents, and stool morphology were detected in MP treated ICR mice, as compared to Vehicle treated group. Also, the gastrointestinal (GI) motility and intestinal length were decreased, while the histopathological structure and cytological structure of the transverse colon were remarkably altered in treated mice. Mice exposed to MP also showed a significant decrease in the GI hormone concentration, muscarinic acetylcholine receptors (mAChRs) expression and their downstream signaling pathway, as well as mucin secretion and transcription of the MUC1, MUC2 and Klf4 genes. Subsequent to MP treatment, concentrations of chloride ion and expressions of its channel (CFTR and CIC-2) were decreased, whereas expressions of AQP3 and 8 for water transportation were downregulated by activation of the MAPK/NF-kB signaling pathway. These regulation on water and chloride transportation were verified in intestinal epithelioid cell line (IEC18) after MP treatment. Conclusion: These results are the first to suggest that oral administration of PS-MP induces chronic constipation through the dysregulation of GI motility, mucin secretion, and chloride ion and water transportation in the transverse colon.