title: PAR-1-dependent and PAR-independent pro-inflammatory signaling in human lung fibroblasts exposed to thrombin.
abstract: Proteinase-activated receptors (PARs) are crucial in orchestrating cellular responses to coagulation proteinases, such as thrombin and FXa. Four PARs have been characterized and have been shown to be differentially expressed in mice and humans and between tissues. We have previously shown that in murine lung fibroblasts, PAR-1 is solely responsible for all cellular responses to thrombin and FXa. In contrast, we report here that in primary human lung fibroblasts (pHLFs), known PARs fail to account for all of the cellular responses to thrombin, in particular in the presence of high, but physiologically achievable concentrations of thrombin. We report that pHLFs secrete CCL2 in a PAR-1-dependent manner at low thrombin concentration (∼0.3 nM). At or above 10 nM thrombin, pharmacological antagonism (RWJ-58259) fails to block thrombin-induced CCL2 release; whereas PAR-1 cleavage-blocking monoclonal antibodies (ATAP2 and WEDE15) only partially inhibit thrombin-induced CCL2 secretion. In addition, activation of PAR-3, PAR-4, and transactivation of either PAR-2 or EGFR were ruled out as being responsible for thrombin-mediated CCL2 secretion at high yet standard concentrations of the proteinase. We further provide evidence that PAR-1-dependent and PAR-independent signaling involves the rapid phosphorylation of ERK, which in turn is absolutely required for thrombin-induced CCL2 secretion at both low and standard concentration of the proteinase. Our findings suggest the existence of a PAR-independent signaling mechanism in human lung fibroblasts and have important implications for the design of therapeutic strategies aimed at blocking pro-inflammatory signaling responses associated with excessive thrombin generation.
authors.affiliation: Centre for Respiratory Research, University College London, United Kingdom.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Ortiz-Stern
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Deng
authors.forename: Natalia
authors.initials: N
authors.lastname: Smoktunowicz
authors.forename: Paul F
authors.initials: PF
authors.lastname: Mercer
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Chambers
mesh_terms: D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D007249:Inflammation / Q000378:metabolism*; D008168:Lung / Q000166:cytology; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D044463:Receptor, PAR-1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000008:administration & dosage / Q000378:metabolism*; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D007191:Indazoles; C424723:RWJ 58259; D044463:Receptor, PAR-1; D014508:Urea; C512478:EGFR protein, human; D066246:ErbB Receptors; D013917:Thrombin; D010975:Platelet Aggregation Inhibitors