James Reihill

Rosiglitazone Stimulates Nitric Oxide Synthesis in Human Aortic Endothelial Cells via AMP-activated Protein Kinase

The thiazolidinedione anti-diabetic drugs increase activation of endothelial nitric-oxide (NO) synthase by phosphorylation at Ser-1177 and increase NO bioavailability, yet the molecular mechanisms that underlie this remain poorly characterized. Several protein kinases, including AMP-activated protein kinase, have been demonstrated to phosphorylate endothelial NO synthase at Ser-1177. In the current study we determined the role of AMP-activated protein kinase in rosiglitazone-stimulated NO synthesis. Stimulation of human aortic endothelial cells with rosiglitazone resulted in the time- and dose-dependent stimulation of AMP-activated protein kinase activity and NO production with concomitant phosphorylation of endothelial NO synthase at Ser-1177. Rosiglitazone stimulated an increase in the ADP/ATP ratio in endothelial cells, and LKB1 was essential for rosiglitazone-stimulated AMPK activity in HeLa cells. Infection of endothelial cells with a virus encoding a dominant negative AMP-activated protein kinase mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production. Furthermore, the stimulation of AMP-activated protein kinase and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated receptor-γ inhibitor GW9662. These studies demonstrate that rosiglitazone is able to acutely stimulate NO synthesis in cultured endothelial cells by an AMP-activated protein kinase-dependent mechanism, likely to be mediated by LKB1.

Inhibition of Protease–Epithelial Sodium Channel Signaling Improves Mucociliary Function in Cystic Fibrosis Airways

RATIONALE In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of the epithelial sodium channel (ENaC) have therapeutic potential in CF airways to reduce hyperstimulated sodium and fluid absorption to levels that can restore airway hydration. OBJECTIVES To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function. METHODS Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy), and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured using a lactate dehydrogenase assay. MEASUREMENTS AND MAIN RESULTS QUB-TL1 inhibits extracellularly located channel activating proteases (CAPs), including prostasin, matriptase, and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells. QUB-TL1-mediated CAP inhibition results in diminished ENaC-mediated Na(+) absorption in CF airway epithelial cells caused by internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A-induced cell death. CONCLUSIONS QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation.

Protein Kinase C phosphorylates AMP-activated protein kinase α1 Ser487

The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues.

A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause disease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consisting of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar compound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aeruginosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.

Trypsin-like protease activity predicts disease severity and patient mortality in adults with cystic fibrosis

Introduction Serine trypsin-like (TL) proteases, which are excessively active in CF airways, promote activation of the epithelial sodium channel (ENaC) and airways dehydration; a key initiating factor for CF lung disease pathogenesis. Furthermore TL-proteases enhance mucin gene expression and mucus hypersecretion, yet whether there is any relationship between the activity of these enzymes and CF pulmonary disease is unknown. Objectives The primary objective of the current investigation was to determine whether TL-protease activity, measured in adult CF sputum sol, correlates with lung disease and patient outcome (survival). A secondary objective was to compare the strength of any relationships observed with that of neutrophil elastase (NE), an established protease biomarker. Methods In this cross sectional retrospective study we analysed CF sputum sol collected from 30 clinically stable adult CF patients. Protease activity was measured by monitoring the hydrolysis of peptide-based substrates. Biomarkers of inflammation (IL-8 and TNF-α) were measured by ELISA. Lung function was assessed by spirometry (FEV1). Mortality data was retrospectively obtained and time in months until death or transplantation used for subsequent survival analysis. Results TL-protease activity inversely correlated with lung function (FEV1) (r = −0.4, p = 0.031) however, no relationship with IL-8 and TNFα was observed. In contrast, NE was found to correlate with IL-8: r = 0.7, p < 0.001 and TNFα: r = 0.7, p < 0.001 but showed no relationship with lung function, indicating that these serine proteases play very distinct roles within the disease process. Kaplan-Meier analysis demonstrated significantly reduced survival for those individuals with above median TL-protease activity. Levels of NE activity showed no relationship with patient survival. Using a multivariate Cox regression analysis (adjusted for age and BMI) a significantly increased mortality hazard (HR 1.028, 95% CI: 1.007–1.049; p = 0.009) was also identified. These findings are supported by analysis of a validation cohort consisting of samples collected from a separate cohort of 33 adult CF patients. Conclusions TL-protease activity inversely correlates with lung function and patient survival. As such tryptic activity may warrant consideration when modelling CF survivorship and should be investigated further as a biomarker of CF lung disease and as a potential therapeutic target.