Mucus overproduction in inflammatory and obstructive airway diseases is associated with goblet cell (GC) metaplasia in airways. Although the mechanisms involved in GC metaplasia and mucus hypersecretion are not completely understood, association with oxidative stress and epidermal growth factor receptor (EGFR) signaling has been reported. To explore the mechanisms involved in oxidative stress-induced GC metaplasia, cultures of differentiated normal human bronchial epithelial cells grown at the air-liquid interface were exposed to reactive oxygen species (ROS) generated by xanthine/xanthine oxidase. EGFR activation and signaling was assessed by measuring EGF and transforming growth factor-alpha release and EGFR and (44/42)MAPK phosphorylation. The GC population was evaluated by confocal microscopy. ROS-induced EGFR activation resulted in GC proliferation and increased MUC5AC gene and protein expression. Signaling was due to pro-EGF processing by tissue kallikrein (TK), which was activated by ROS-induced hyaluronan breakdown. It was inhibited by catalase, a TK inhibitor, and EGF-blocking antibodies. Exposure to recombinant TK mimicked the ROS effects, increasing the expression of MUC5AC and lactoperoxidase. In addition, ROS induced the antiapoptotic factor Bcl-2 in a TK-dependent fashion. In conclusion, ROS-induced GC metaplasia in normal human bronchial epithelial cells is associated with HA depolymerization and EGF processing by TK followed by EGFR signaling, suggesting that increases in TK activity could contribute to GC metaplasia and mucus hypersecretion in diseases such as asthma and chronic bronchitis. The data also suggest that increases in GC population could be sustained by the associated upregulation of Bcl-2 in airway epithelial cells.

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALI), treated with PI-specific phospholipase C (PI-PLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported that HA degradation was mediated by reactive oxygen species (ROS) in human airways. Here we show that ROS increase Hyal2 expression and activity in NHBE cells and that the p38MAPK signaling pathway is involved in this effect. Hyal2 induction was confirmed by using small interfering RNA (siRNA) expressing lentivirus. These in vitro findings correlated in vivo with smokers, where increased Hyal2 immunoreactivity in the epithelium was associated with augmented levels of HA and the appearance of low molecular mass HA species in bronchial secretions. In summary, this work provides evidence that ROS induce Hyal2, suggesting that Hyal2 is likely responsible for the sustained HA fragmentation in the airway lumen observed in inflammatory conditions associated with oxidative stress. Hyaluronan (HA)3 is a non-sulfated glycosaminoglycan found in the extracellular matrix, in body fluids, and in secretions of mammals. HA is synthesized by three transmembrane isoenzymes: HAS1, HAS2, and HAS3 at the inner face of the plasma membrane and translocated into the extracellular space (1).The association with various binding proteins (2, 3) confers HA with a unique plasticity to organize extracellular matrix (ECM) in a tissue specific fashion (for review see Ref. 4), varying from a tightly cross-linked mesh in cartilage to a highly hydrated matrix in dermis and vitreous humor (5).In addition, HA induces intracellular signaling by binding specific receptors at the cell surface, (6, 7) orchestrating a variety of host responses generally requiring an extensive deposition of a HA in the ECM. This deposition is essential for cumulus oophorus fertilization (8) as well as for proliferation and migration of mesenchymal cells. Airway smooth muscle cells exposed to polyinosinic acid-polycytidylic acid synthesize an abnormal HA matrix with cable-like structures that bind and retains leukocytes (9, 10), suggesting that HA play key roles on host defense against infections as well.Biological functions of HA are associated with its size (6, 11). For instance, high molecular weight HA (HMWHA) exhibit anti-angiogenic, anti-inflammatory, and immunosuppressive effects while small HA fragments are angiogenic and proinflammatory (12, 13). The contrasting responses elicited by different sizes of HA are exemplified in a recent report in which HMWHA attenuated while low molecular weight HA (LMWHA) increased ozone-induced airway hyperreactivity, in a mouse model of asthma (14).In human airway epithelium, HA is present at the lumen as...

In human airways, oxidative stress-induced submucosal gland cell hypertrophy and hyperplasia, histological features of chronic bronchitis, have been linked to epidermal growth factor receptor (EGFR) activation. To explore mechanisms of oxidative stress-induced EGFR activation and signaling, primary cultures of human tracheal submucosal gland (SMG) cells were used to assess EGFR ligand release, EGFR phosphorylation, p44/42 MAPK phosphorylation, and mucin 5AC synthesis in response to reactive oxygen species generated by xanthine/xanthine oxidase (X/XO). Exposure to X/XO increased release of epidermal growth factor (EGF) from these cells, thereby activating EGFR, phosphorylating MAPK, and increasing mucin 5AC production. The importance of EGF was confirmed by transfection of small interfering RNA inhibiting pro-EGF production, which resulted in inhibition of EGFR and MAPK phosphorylation despite X/XO exposure. Blocking signaling by using specific protease inhibitors showed that tissue kallikrein (TK) processed pro-EGF in response to X/XO. Airway TK is bound and inactivated by luminal hyaluronan (HA), and treatment of submucosal gland cells with X/XO induced HA depolymerization and TK activation. These events were blocked by reactive oxygen species scavengers and addition of exogenous excess HA and TK inhibitors. Thus, HA plays a crucial role in regulating airway TK activity and thereby TK-mediated release of active EGF from human SMG cells. Sustained HA depolymerization is expected to cause TK activation, EGF release, and EGFR signaling and to lead to SMG cell hypertrophy and hyperplasia as well as mucus hypersecretion with subsequent airflow obstruction.

TSG-6 (the protein product of TNF-stimulated gene-6), an inflammation-associated protein, forms covalent complexes with heavy chains (HCs) from inter-␣-inhibitor and pre-␣-inhibitor and associates noncovalently with their common bikunin chain, potentiating the antiplasmin activity of this serine protease inhibitor. We show that TSG-6 and TSG-6·HC complexes are present in bronchoalveolar lavage fluid from patients with asthma and increase after allergen challenge. Immunodetection demonstrated elevated TSG-6 in the airway tissue and secretions of smokers. Experiments conducted in vitro with purified components revealed that bikunin·HC complexes (byproducts of TSG-6·HC formation) release bikunin. Immunoprecipitation revealed that bikunin accounts for a significant proportion of tissue kallikrein inhibition in bronchoalveolar lavage after allergen challenge but not in baseline conditions, confirming that bikunin in its free state, but not when associated with HCs, is a relevant protease inhibitor in airway secretions. In primary cultures of differentiated human airway epithelial and submucosal gland cells, TSG-6 is induced by TNF-␣ and IL-1␤, which suggests that these cells are responsible for TSG-6 release in vivo. Bikunin and HC3 (i.e., pre-␣-inhibitor) were also induced by TNF-␣ in primary cultures. Our results suggest that TSG-6 may play an important protective role in bronchial epithelium by increasing the antiprotease screen on the airway lumen.

Background: Cigarette smoke (CigS) induces hyaluronan fragmentation and increases epithelial permeability. Results: CigS and HA fragments decrease E-cadherin expression that is prevented by knocking down layilin. Conclusion: HA fragments bind to layilin and signal through RhoA/ROCK to inhibit E-cadherin. Significance: Airway epithelium is our first line of defense against inhaled insults. HA fragments released by CigS disrupt this barrier.

Mucus hypersecretion with elevated MUC5B mucin production is a pathologic feature in many airway diseases associated with oxidative stress. In the present work, we evaluated MUC5B expression in airways and in primary cultures of normal human bronchial epithelial (NHBE) cells, as well as the mechanisms involved in its regulation. We found that oxidative stress generated by cigarette smoke or reactive oxygen species (ROS) induces MUC5B up-regulation in airway epithelium from smokers and in NHBE cells, respectively. We have previously shown that ROS-induced MUC5AC expression in NHBE cells is dependent on hyaluronan depolymerization and epidermal growth factor receptor (EGFR)/mitogenactivated protein kinase (MAPK) activation. Since hyaluronan fragments can activate MAPK through the hyaluronan receptor CD44, and CD44 heterodimerizes with EGFR, we tested whether ROS and/ or hyaluronan fragments induce MUC5B mRNA and protein expression through CD44/EGFR. We found that ROS promotes CD44/EGFR interaction, EGFR/MAPK activation, and MUC5B up-regulation that are prevented by blocking CD44 and/or EGFR. These results were mimicked by hyaluronan fragments. In summary, our results show that oxidative stress in vivo (cigarette smoke) or in vitro (ROS) induces MUC5B up-regulation. This ROS-induced MUC5B expression requires CD44 as well as EGFR and MAPK activation. In addition, we also provide evidence that hyaluronan fragments are sufficient to induce CD44/EGFR interaction and downstream signaling that results in MUC5B up-regulation, suggesting that hyaluronan depolymerization during inflammatory responses could be directly involved in the induction of mucus hypersecretion.Keywords: MUC5B; hyaluronan fragments; CD44; airway epithelium Mucus overproduction occurs in a variety of acute reactions to cigarette smoke (1) and microorganisms (2, 3), as well as in chronic airway inflammatory diseases such as chronic bronchitis (4-6), asthma (7-9), bronchiectasis (10), and cystic fibrosis (11). The major macromolecular components of mucus are large and heavily glycosylated mucin proteins that are encoded by various MUC genes (12)(13)(14), that maintain airways homeostasis by protecting the epithelial surface from environmental insults. MUC5B and MUC5AC are the major mucins present in airways secretions from patients with asthma (15-17), cystic fibrosis (18,19), and chronic obstructive pulmonary disease (COPD) (1). In normal airways, MUC5AC and MUC5B are typically described as produced by goblet cells at the surface epithelium (20) and by mucous cells in submucosal glands (21), respectively. However, MUC5B mucin is also present at low levels in airway epithelium from normal lung donors (22) and up-regulated in patients with airways diseases and inflammation (1,(23)(24)(25)(26)(27). Nevertheless, it is not clear whether MUC5B and MUC5AC are produced by the same or by different cell populations in airway epithelium, since co-localization studies have not been done. Compared with MUC5AC expression regulation, the pathways involved in the regu...

Glycosaminoglycans (GAGs), known to be present in airway mucus, are macromolecules with a variety of structural and biological functions. In the present work, we used fluorophore-assisted carbohydrate electrophoresis (FACE) to identify and relatively quantify GAGs in human tracheal aspirates (HTA) obtained from healthy volunteers. Primary cultures of normal human bronchial epithelial (NHBE) and submucosal gland (SMG) cells were used to assess their differential contribution to GAGs in mucus. Distribution was further assessed by immunofluorescence in human trachea tissue sections and in cell cultures. HTA samples contained keratan sulfate (KS), chondroitin/dermatan sulfate (CS/DS), and hyaluronan (HA), whereas heparan sulfate (HS) was not detected. SMG cultures secreted CS/DS and HA, CS/DS being the most abundant GAGs in these cultures. NHBE cells synthesized KS, HA, and CS/DS. Confocal microscopy showed that KS was exclusively found at the apical border of NHBE cells and on the apical surface of ciliated epithelial cells in tracheal tissues. CS/DS and HA were present in both NHBE and SMG cells. HS was only found in the extracellular matrix in trachea tissue sections. In summary, HTA samples contain KS, CS/DS, and HA, mirroring a mixture of secretions originated in surface epithelial cells and SMGs. We conclude that surface epithelium is responsible for most HA and all KS present in secretions, whereas glands secrete most of CS/DS. These data suggest that, in diseases where the contribution to secretions of glands versus epithelial cells is altered, the relative concentration of individual GAGs, and therefore their biological activities, will also be affected.

Hypercapnia, the elevation of CO2 in blood and tissue, commonly develops in patients with advanced lung disease and severe pulmonary infections, and is associated with high mortality. We previously reported that hypercapnia alters expression of host defense genes, inhibits phagocytosis, and increases the mortality of Pseudomonas pneumonia in mice. However, the effect of hypercapnia on autophagy, a conserved process by which cells sequester and degrade proteins and damaged organelles that also plays a key role in antimicrobial host defense and pathogen clearance, has not previously been examined. In the present study we show that hypercapnia inhibits autophagy induced by starvation, rapamycin, LPS, heat-killed and live bacteria in the human macrophage. Inhibition of autophagy by elevated CO2 was not attributable to acidosis. Hypercapnia also reduced macrophage killing of Pseudomonas aeruginosa. Moreover, elevated CO2 induced the expression of Bcl-2 and Bcl-xL, anti-apoptotic factors that negatively regulate autophagy by blocking Beclin 1, an essential component of the autophagy initiation complex. Furthermore, siRNA targeting Bcl-2 and Bcl-xL and the small molecule Z36, which blocks Bcl-2 and Bcl-xL binding to Beclin 1, prevented hypercapnic inhibition of autophagy and bacterial killing. These results suggest that targeting the Bcl-2/Bcl-xL-Beclin 1 interaction may hold promise for ameliorating hypercapnia-induced immunosuppression and improving resistance to infection in patients with advanced lung disease and hypercapnia.