Abstract
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.
Highlights
We have recently shown that Hyals 1, 2, and 3 are expressed in airway epithelium as well as in Normal human bronchial epithelial cells (NHBE) cells grown at the air-liquid interphase (ALI)
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 Hyaluronidase 2 (Hyal2)-small interfering RNA (siRNA) and non-insert controls exposed to reactive oxygen species (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 of 1 mM EDTA, 0.2% Triton X-100 sodium formate at 37 °C for 24 h
Hyal3 (1.13 Ϯ 0.05; 1.23 Ϯ 0.11-fold, respectively) expression was ROS Exposure Induces Hyal Activity and Hyal2 mRNA in NHBE Cells—We have previously shown that Hyal1, 2, and 3 are expressed in bronchial epithelium, and that Hyal2 was present at the apical membrane of airway epithelium [26]
Summary
Isolation Procedure—Human tracheas and main bronchi, obtained from donor lungs through the University of Miami Life Alliance Organ Recovery Agency with approval from the local Institutional Review Board (IRB) were opened at the membranous portion, and the mucosa was dissected off the cartilage. In studies aimed at confirming that Hyal was attached to the cell membrane by a GPI anchor, NHBE cultures were treated apically with phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus (Invitrogen) in HEPES buffer pH 7.4 for 30 min at 37 °C. RNA was extracted from NHBE cultures using Trizol reagent (Invitrogen) and reverse transcribed into cDNA using iScriptTM cDNA synthesis kit (Bio-Rad) according to manufacturer’s instructions. Aliquots of cells lysates containing equal amounts of protein (n ϭ 3 different lung donors) were run on 4 –15% TrisHCl Ready gels (Bio-Rad) and transferred to polyvinylidene fluoride (PVDF, Millipore, Billerica, MA). Cell lysate aliquots containing 25 g of protein were run on 4 –15% Tris-HCl Ready Gels (Bio-Rad) and transferred to PVDF membranes Visualization of Hyal was achieved using the mouse anti-Hyal polyclonal antibody (1/500) followed by alkaline phosphatase-conjugated antimouse antibodies (1/5,000 (v/v); KPL). Differentiated cells determined by the presence of mucus secretion and beating cilia (21 days on air) were used for the experiments
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
