Date Published: February 22, 2018
Publisher: Public Library of Science
Author(s): Hyo Jung Sim, Sang-Hyun Kim, Kyung-Jae Myung, Taejoon Kwon, Hyun-Shik Lee, Tae Joo Park, Wei-Chun Chin.
The airway epithelium in human plays a central role as the first line of defense against environmental contaminants. Most respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and respiratory infections, disturb normal muco-ciliary functions by stimulating the hypersecretion of mucus. Several muco-active agents have been used to treat hypersecretion symptoms in patients. Current muco-active reagents control mucus secretion by modulating either airway inflammation, cholinergic parasympathetic nerve activities or by reducing the viscosity by cleaving crosslinking in mucin and digesting DNAs in mucus. However, none of the current medication regulates mucus secretion by directly targeting airway goblet cells. The major hurdle for screening potential muco-active agents that directly affect the goblet cells, is the unavailability of in vivo model systems suitable for high-throughput screening. In this study, we developed a high-throughput in vivo model system for identifying muco-active reagents using Xenopus laevis embryos. We tested mucus secretion under various conditions and developed a screening strategy to identify potential muco-regulators. Using this novel screening technique, we identified narasin as a potential muco-regulator. Narasin treatment of developing Xenopus embryos significantly reduced mucus secretion. Furthermore, the human lung epithelial cell line, Calu-3, responded similarly to narasin treatment, validating our technique for discovering muco-active reagents.
The muco-ciliary epithelium of the airway tract is one of the most vulnerable tissues which are constantly challenged by external pathogens or contaminants. The airway epithelium consists of several cell types, such as the mucus secreting goblet cells, multi-ciliated cells, and basal cells . The goblet cells synthesize and secrete mucus components such as mucin protein (Muc5AC or Muc5B in mammals), whereas the cilia of the motile ciliated cells expectorate the mucoid fluid, and the basal cells possibly possess a differentiation potential . Mucus hypersecretion is one of the major symptoms of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and respiratory infections . Most medications for reducing mucus hypersecretion either alleviate inflammation, indirectly regulate mucus secretion as in the case of steroids, or reduce the viscosity of mucus to facilitate expectoration by reducing the disulfide bond of mucin as in the case of N-acetylcysteine or by digesting DNAs in the mucus as in the case of dornase . Current experimental models for studying the activities of muco-regulators, including mouse tracheal primary cultures and human bronchial epithelial culture, are excellent for investigating the pathophysiology of mucus production and secretion. However, these techniques are labor, resource, and time-intensive . Thus, current experimental models are not adequate for unbiased high throughput screening-based and unbiased drug discovery.
In this study, we exploited the advantages of the Xenopus embryonic epithelium to develop an unbiased drug discovery strategy for novel muco-active reagents. Indeed, our novel technique efficiently and effectively detected changes in mucus levels in the nourishing media by a simple two-step analysis procedure involving WGA-HRP detection and chromogenic measurement. Furthermore, we tested the muco-regulatory activities of a natural compound library to validate our high throughput screening procedure, which is currently challenging in other popular model systems such as the HBEC ALI culture or mouse models. We have identified several muco-active reagents from this pioneering natural compound library screening. Among several candidates, two chemicals, phorbol 12,13-dibutyrate and bicuculline, are known to control mucus secretion as a PKC activator and GABA receptor antagonist respectively. These data validated the effectiveness of our technique. In addition, we identified a novel muco-active compound called narasin, which can possibly be used as a therapeutic for respiratory diseases. Genomic analysis in Xenopus tropicalis, revealed a huge expansion of Muc genes in its genome . Apparently, Xenopus genome contains 26 gel forming mucin genes including 12 Muc2 genes (Muc2A-L), and 11 Muc5 genes (Muc5A-K). Although, the Muc genes in Xenopus are highly evolved and differ structurally from those of humans, our data suggest the secretory mechanism may be conserved in Xenopus and can be used to discover the muco-regulator affecting mucus secretion from the goblet cells. Further research on the physiology and molecular mechanism of Muc gene regulation in Xenopus would strengthen the utility of embryonic epidermis as an alternative model system to study mucociliary epithelium.