Deng, X.; (2008) Identification of PAR1-G protein signaling pathways involved in thrombin-induced CCL2 MCP-1 production. Doctoral thesis , University of London.

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Abstract

Uncontrolled activation of the coagulation cascade following lung injury has been implicated in both lung inflammation and fibrosis. In addition to its role in coagulation, thrombin exerts pluripotent cellular effects via the activation of its high-affinity receptor, proteinase activated receptor-1 (PARi). PARi is a seven transmembrane domain G protein-coupled receptor that exhibits the ability to couple to multiple G protein family subunits, including Gaj/Ql Gaq and Gcfi2/13 within the same cell type. Activation of PAR on fibroblasts, a key effector cell in lung fibrosis, results in the induction of several mediators, including the potent monocyte and fibrocyte chemoattractant CCL2. In this thesis, the G-protein and downstream signalling pathways involved in PARr mediated CCL2 production and release were examined. Using a novel PARt antagonist which blocks the interaction between PARi and Gaq, this thesis shows for the first time that PARt coupling to Gaq is essential for thrombin (10nM)-induced CCL2 gene expression and protein release in murine lung fibroblasts (MLFs). The work presented here further demonstrates that these effects are mediated via the cooperation between ERK1/2 and Rho kinase signalling pathways: a calcium-independent PKC, c-Raf and ERK1/2 pathway was found to mediate PARr-induced CCL2 gene transcription whereas PLC, calcium, calcium-dependent PKC and Rho kinase pathway influences CCL2 protein release. This thesis represents the first demonstration of the cooperation between two pathways in mediating the stimulatory effects of thrombin, or indeed any other extracellular stimulus, on the induction and release of the potent chemoattractant, CCL2. This thesis also examined the signalling receptor and downstream effectors involved in thrombin-induced CCL2 production in primary human lung fibroblasts (pHALFs). The results demonstrate that PARi coupling to Gaq is also both necessary and sufficient in mediating thrombin-induced CCL2 production at low concentration of the proteinase, whereas at high concentrations, these effects may be partially PAR-independent. This discrepancy between MLFs and pHALFs may be explained by differences of PAR receptor expression between species. Taken together, this thesis proposes that targeting the interaction between PARi and specific G proteins may allow more selective blockade of PARi pro-inflammatory and pro-fibrotic signalling, whilst preserving the essential role of other PAR-mediated cellular responses.

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