PAR2-induced inflammatory responses in human kidney tubular epithelial cells

Vesey DA, Suen JY, Seow V, Lohman RJ, Liu L, Gobe GC, Johnson DW, Fairlie DP. PAR2-induced inflammatory responses in human kidney tubular epithelial cells. Am J Physiol Renal Physiol 304: F737-F750, 2013. First published January 2, 2013; doi:10.1152/ajprenal.00540.2012.-Protease-activated receptor-2 (PAR2) is a G protein-coupled receptor abundantly expressed in the kidney. The aim of this study was to profile inflammatory gene and protein expression induced by PAR2 activation in human kidney tubular epithelial cells (HTEC). A novel PAR2 antagonist, GB88, was used to confirm agonist specificity. Intracellular Ca2+ (iCa(2+)) mobilization, confocal microscopy, gene expression profiling, qRTPCR, and protein expression were used to characterize PAR2 activation. PAR2 induced a pronounced increase in iCa(2+) concentration that was blocked by the PAR2 antagonist. Treatment with SLIGKV-NH2 at the apical or basolateral cell surface for 5 h induced expression of a range of inflammatory genes by greater than fourfold, including IL-1 beta, TRAF1, IL-6, and MMP-1, as assessed by cDNA microarray and qRTPCR analysis. Using antibody arrays, GM-CSF, ICAM-1, TNF-alpha, MMP-1, and MMP-10 were among the induced proteins secreted. Cytokine-specific ELISAs identified three-to sixfold increases in GM-CSF, IL-6, IL-8, and TNF-alpha, which were blocked by GB88 and protein kinase C inhibitors. Treatment of cells at the basolateral surface induced more potent inflammatory responses, with release of MCP-1 and fibronectin to the apical and basolateral compartments; apical treatment only increased secretion of these factors to the apical compartment. PAR2 activation at the basolateral surface dramatically reduced transepithelial electrical resistance (TEER) whereas apical treatment had no effect. There was very little leakage