Inflammatory mediators modulate thrombin and cathepsin-G signaling in human bronchial fibroblasts by inducing expression of proteinase-activated receptor-4

Human lung fibroblasts express proteinase-activated receptor-1 (PAR(1)), PAR(2) and PAR(3), but not PAR(4). Because PAR(2) has inflammatory effects on human primary bronchial fibroblasts (HPBF), we asked 1) whether the inflammatory mediators TNF-alpha and LPS could modify HPBF PAR expression and 2) whether modified PAR expression altered HPBF responsiveness to PAR agonists in terms of calcium signaling and cell growth. TNF-alpha and LPS induced PAR(4) mRNA expression (RT-PCR) at 6 h and 24 h, respectively. TNF-alpha and LPS also upregulated PAR(2) mRNA expression with similar kinetics but had negligible effect on PAR(1) and PAR(3). Flow cytometry for PAR(1), PAR(2), and PAR(3) also demonstrated selective PAR(2) upregulation in response to TNF-alpha and LPS. Intracellular calcium signaling to SLIGKV-NH2 (a selective PAR(2)-activating peptide; PAR(2)-AP) and AYPGQV-NH2 (PAR(4)-AP) revealed that TNF-alpha and LPS induced maximal responses to these PAR agonists at 24 h and 48 h, respectively. Upregulation of PAR(2) by TNF-alpha heightened HPBF responses to trypsin, while PAR(4) induction enabled cathepsin-G-mediated calcium signaling. Cathepsin-G also disarmed PAR(1) and PAR(2) in HPBF, while tryptase disarmed PAR(2). Induction of PAR(4) also enabled thrombin to elicit a calcium signal through both PAR(1) and PAR(4), as determined by a desensitization assay. In cell growth assays the PAR(4) agonists cathepsin-G and AYPGQV-NH2 reduced HPBF cell number only in TNF-alpha-treated HPBF. Moreover, the mitogenic effect of thrombin (a PAR(1)/PAR(4) agonist) but not the PAR(1)-AP TFLLR-NH2, was ablated in TNF-alpha-treated HPBF. These findings point to an important mechanism, whereby cellular responses to thrombin and cathepsin-G can be modified during an inflammatory response.