Abstract
Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI-H520 squamous cells increased intracellular calcium levels and granulocyte macrophage-colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air-liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.
Highlights
Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi
We utilized a fluorogenic assay (Trio assay) to directly visualize Protease-activated receptors (PARs)-2 activation by its interaction with -arrestin [26], which associates with activated and phosphorylated G-protein– coupled receptors (GPCRs) and mediates further signal transduction and/or internalization to endosomes (Fig. 1a) [27]. This assay is based on a tripartite GFP, with the 11th -strand of GFP fused to the C terminus of PAR-2, the 10th -strand of GFP fused to -arrestin, and -strands 1–9 of GFP expressed as a soluble protein [26]
Prior literature on PAR-2 in the airway has focused on increased PAR-2 expression in inflamed airway mucosa [2, 38, 47]
Summary
Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. We previously found that PAR-2 is expressed on the basolateral membrane of healthy and wellpolarized differentiated primary sinonasal epithelial ciliated cells, where it elevates intracellular calcium to increase ciliary beating and apical membrane ClϪ secretion [8], suggesting PAR-2 promotes epithelial mucociliary clearance in response to protease activity. We hypothesized basolateral polarization of PAR-2 may limit its activation except during times of epithelial barrier breakdown during infection or inflammation. Much of the literature on airway remodeling focuses on barrier dysfunction, but airway cells in vitro can maintain tight
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