Vanadium pentoxide (V(2)O(5)) induced mucin production by airway epithelium.

Exposure to environmental pollutants has been linked to various airway diseases and disease exacerbations. Almost all chronic airway diseases such as chronic obstructive pulmonary disease and asthma are caused by complicated interactions between gene and environment. One of the major hallmarks of those diseases is airway mucus overproduction (MO). Excessive mucus causes airway obstruction and significantly increases morbidity and mortality. Metals are major components of environmental particulate matters (PM). Among them, vanadium has been suggested to play an important role in PM-induced mucin production. Vanadium pentoxide (V(2)O(5)) is the most common commercial source of vanadium, and it has been associated with occupational chronic bronchitis and asthma, both of which are MO diseases. However, the underlying mechanism is not entirely clear. In this study, we used both in vitro and in vivo models to demonstrate the robust inductions of mucin production by V(2)O(5). Furthermore, the follow-up mechanistic study revealed a novel v-raf-1 murine leukemia viral oncogene homolog 1-IKK-NF-κB pathway that mediated V(2)O(5)-induced mucin production. Most interestingly, the reactive oxygen species and the classical mucin-inducing epidermal growth factor receptor (EGFR)-MAPK pathway appeared not to be involved in this process. Thus the V(2)O(5)-induced mucin production may represent a novel EGFR-MAPK-independent and environmental toxicant-associated MO model. Complete elucidation of the signaling pathway in this model will not only facilitate the development of the treatment for V(2)O(5)-associated occupational diseases but also advance our understanding on the EGFR-independent mucin production in other chronic airway diseases.

Rhinovirus-induced major airway mucin production involves a novel TLR3-EGFR-dependent pathway.

Mucociliary clearance is a critical innate defense system responsible for clearing up invading pathogens including bacteria and virus. Although the right amount of mucus is good, excessive mucus causes airway obstruction and tends to precipitate disease symptoms. Rhinovirus (RV) is a common cold virus that causes asthma and chronic obstructive pulmonary disease exacerbation. Mucus overproduction has been linked to the pathogenesis of RV-induced diseases and disease exacerbations. However, the molecular mechanism is not clear. In this study, using one of the major airway mucin-MUC5AC as marker, we found that both major and minor groups of RV induced mucin production in primary human epithelial cells and cell line. RV1A (a minor group of RV) could induce mucous cell metaplasia in vivo. Viral replication was needed for RV-induced mucin expression, and this induction was also dependent on TLR3, suggesting the involvement of double-stranded (ds) RNA signaling. Indeed, dsRNA alone could also induce mucin expression. TLR3-mediated mucin induction was negatively regulated by MyD88, and only partially dependent on TRIF, which suggests a departure from well-documented TLR3 signaling paradigm that mediates inflammatory and other innate defense gene inductions. In addition, TLR3 signaling activated epidermal growth factor receptor (EGFR) through inductions of the expression of EGFR ligands (transforming growth factor-alpha and amphiregulin), which in turn activated EGFR-ERK signaling and mucin expression through an autocrine/paracrine loop. This novel coupling of antiviral defense machinery (i.e., TLR3) and major epithelial proliferation/repair pathway (i.e., EGFR) might play an important role in viral-induced airway remodeling and airway disease exacerbation.

TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

Rhinovirus induces airway epithelial gene expression through double-stranded RNA and IFN-dependent pathways.

Rhinovirus (RV) infection is the major cause of common colds and of asthma exacerbations. Because the epithelial cell layer is the primary target of RV infection, we hypothesize that RV-induced airway disease is associated with the perturbation of airway epithelial gene expression. In this study, well differentiated primary human airway epithelial cells were infected with either RV16 (major group) or RV1B (minor group). Transcriptional gene profiles from RV-infected and mock-infected control cells were analyzed by Affymetrix Genechip, and changes of the gene expression were confirmed by real-time RT-PCR analysis. At 24 h after infection, 48 genes induced by both viruses were identified. Most of these genes are related to the IFN pathway, and have been documented to have antiviral functions. Indeed, a significant stimulation of IFN-beta secretion was detected after RV16 infection. Neutralizing antibody specific to IFN-beta and a specific inhibitor of the Janus kinase pathway both significantly blocked the induction of RV-inducible genes. Further studies demonstrated that 2-aminopurine, a specific inhibitor double-stranded RNA-dependent protein kinase, could block both IFN-beta production and RV-induced gene expression. Thus, IFN-beta-dependent pathway is a part of the double-stranded RNA-initiated pathway that is responsible for RV-induced gene expression. Consistent with its indispensable role in the induction of antiviral genes, deactivation of this signaling pathway significantly enhanced viral production. Because increase of viral yield is associated with the severity of RV-induced airway illness, the discovery of an epithelial antiviral signaling pathway in this study will contribute to our understanding of the pathogenesis of RV-induced colds and asthma exacerbations.