Bronchial Tissue Kallikrein Activity Is Regulated by Hyaluronic Acid Binding

Abstract

Tissue kallikrein (TK) is secreted by serous cells of tracheobronchial submucosal glands and plays a role in allergic airway responses. To better understand the regulation of TK, we used primary cultures of submucosal gland cells that release TK upon stimulation. Media from cultures stimulated with chymase (10(-7) M) showed increased TK activity (0.50 +/- 0.22 mU/ml mean +/- standard error) in comparison with the control group (0.08 +/- 0.02 mU/ml). The increased TK activity was significantly correlated with increases in the levels of the serous cell marker, secretory leukoprotease inhibitor. Anion exchange chromatography of the conditioned culture media showed that TK activity eluted as a broad peak between 1.6 and 1.8 M NaCl, unlike the reported elution (0.3 to 0.6 M NaCl) of kallikreins from other tissues, suggesting that secreted bronchial TK was bound to a negatively charged molecule. Hyaluronidase digestion increased TK activity in both pre- and post-chymase-stimulated culture media, whereas no such change was seen after samples were digested with heparinase or chondroitinase ABC. Further, after hyaluronidase digestion of media, TK eluted from an anion exchange column between 0.3 and 0.6 M NaCl. Enzymatic detection of TK after nondenaturing gel electrophoresis showed that hyaluronidase digestion also reduced the electrophoretic heterogeneity of TK to a single band, whereas adding back hyaluronic acid (HA) to hyaluronidase-digested samples restored the original heterogeneity. Finally, TK activity bound to HA-Sepharose and could be eluted with HA. These studies show that primary cultures of ovine submucosal gland cells secrete TK in a regulated fashion, and that secreted TK binds to HA. This binding reduces TK enzymatic activity; therefore, factors that affect HA turnover could modify the TK activity in the airway lumen. These events could be important in the regulation of kinin-mediated airway inflammation.

Hyaluronan and Layilin Mediate Loss of Airway Epithelial Barrier Function Induced by Cigarette Smoke by Decreasing E-cadherin

“…To confirm that the layilin used in our experiments binds to hyaluronan as reported previously (39), 0.8 g of layilin (Novus, Littleton, CO) was added to HA-Sepharose prepared as described (51,52) and incubated for 1 h at RT. After extensive washing with 0.5 M NaCl in PBS, beads were eluted with 1 mg/ml HA (35 kDa, R&D Systems).…”

TSG-6 Potentiates the Antitissue Kallikrein Activity of Inter–α-inhibitor through Bikunin Release

“…The enzymatic activity of TK, including its kinetic constants, was determined using a chromogenic substrate as described previously (12,51). Briefly, triplicate samples containing rTK (10 nM) were incubated in an ultralow-binding 96-well plate in the presence of increasing concentrations of I␣I (50 nM to 50 M) and TSG-6Q (750 nM to 750 M) individually or preincubated for 30 min at 37ЊC (to generate TSG-6·HC complexes) in 20 mM HEPES-HCl (pH 7.5) 150 mM NaCl, 5 mM MgCl 2 , and 0.02% (vol/vol) Tween-20 for 2 h at 37ЊC (final volume 100 l).…”

“…SMG cells were grown as described (12,14). After 10 d of growth, cultures showed positive labeling with antibodies against TK and lysozyme (specific serous cell products) and Muc 5AC (specific mucous cell product) that accounted for 89 Ϯ 18% of cells (n ϭ 5 cultures, 100 cells counted per well; mean Ϯ SEM).…”

“…In normal conditions, TK is bound and inhibited by hyaluronan (HA) (12), a nonsulfated glycosaminoglycan present in the apical pole of airway epithelium (13). Depolymerization of HA by ROS or hyaluronidase results in the release and activation of TK (14), a finding further supported by the ability of aerosoldelivered HA in preventing ROS and TK-mediated responses (15,16).…”

Translational Success Stories: Angiotensin Receptor 1 Antagonists in Heart Failure

“…44,45 In addition to an Ang II-forming capacity that is 20-fold higher than ACE, 44 chymase was reported to directly activate matrix metalloproteinases 115,116 and degrade fibronectin, 117 resulting in cell death through the loss of cell-extracellular matrix connections. 118 In addition, chymase was shown to activate kallikrein, 119 transforming growth factor-␤, and IL-1␤, 120 and recently was reported to convert Ang-(1-12) to Ang II. 112,113 In addition to other mast cell contents, the multiple actions of chymase on tissue remodeling were reported to be associated with the pathophysiology of plaque rupture and abdominal aortic aneurysm in humans.…”