Role of Hyaluronan and Reactive Oxygen Species in Tissue Kallikrein-mediated Epidermal Growth Factor Receptor Activation in Human Airways

Abstract

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.

Reactive Oxygen Species and Hyaluronidase 2 Regulate Airway Epithelial Hyaluronan Fragmentation

“…We have recently shown that Hyals 1, 2, and 3 are expressed in airway epithelium as well as in NHBE cells grown at the ALI. We found that Hyal protein expression is increased in the epithelium of asthmatics (26), suggesting that HA fragmentation during inflammatory responses could be due, in addition to ROS (15,21) to Hyal. In in vitro assays in cell-free conditions, ROS formation is fast and…”

“…We have previously reported that ROS induce HA fragmentation (21,23), and that Hyal1, 2, and 3 are expressed in the airway epithelium (26). We and others (26,47) have shown that Hyal2 is selectively increased in inflammatory conditions and that TNF-␣ and IL-1␤ induce Hyal2 gene and protein expression in NHBE cells (26).…”

“…To confirm that the observed increases in Hyal activity correlated with HA degradation, cultures were assessed as previously described (39) with slight modifications: Samples from Hyal2-siRNA and non-insert controls exposed to ROS (n ϭ 3) were lysed and volumes containing equal amount of proteins incubated with 50 g of HMW HA from Streptococcus zooepidemicus in 200 l (final volume) of 1 mM EDTA, 0.2% Triton X-100 sodium formate (pH 4.2) at 37°C for 24 h. After exposure, HA was electrophoresed in agarose gel as described before (15,21,40), mobility changes were assessed using Select-HA TM standards (Hyalose, L.L.C., Oklahoma City OK) and visualized using Stains-All.…”

“…It is released by submucosal glands and by superficial airway epithelial cells (16,17) where it serves as an anchor for secreted proteins involved in host defense, preventing their removal by mucociliary clearance (18 -20). Tissue kallikrein (TK), a serine protease involved in airway inflammation and mucus hypersecretion (15,21) is bound to, and inhibited by HMWHA at the airway epithelial surface. Exposure to reactive oxygen species (ROS) depolymerizes HA resulting in the release of active TK (15,21) and HA fragments that stimulate ciliary beating (22) and induce MUC5B up-regulation (23).…”

“…Tissue kallikrein (TK), a serine protease involved in airway inflammation and mucus hypersecretion (15,21) is bound to, and inhibited by HMWHA at the airway epithelial surface. Exposure to reactive oxygen species (ROS) depolymerizes HA resulting in the release of active TK (15,21) and HA fragments that stimulate ciliary beating (22) and induce MUC5B up-regulation (23). Thus, fragmentation of HA at the epithelial surface can profoundly affect cell responses.…”