Dataset Information

Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Galphaq and cooperation between ERK1/2 and Rho kinase signaling pathways.

ABSTRACT: Uncontrolled activation of the coagulation cascade after tissue injury has been implicated in both inflammation and tissue fibrosis. Thrombin exerts pluripotent cellular effects via its high-affinity receptor, proteinase-activated receptor-1 (PAR(1)) and signaling via Galpha(i/o), Galpha(q), or Galpha(12/13). Activation of PAR(1) on fibroblasts, a key effector cell in fibrosis, results in the induction of several mediators, including the potent monocyte and fibrocyte chemoattractant CCL2. The aim of this study was to identify the G protein and signaling pathway involved in PAR(1)-mediated CCL2 production and release. Using a novel PAR(1) antagonist that blocks the interaction between PAR(1) and Galpha(q), we report for the first time that PAR(1) coupling to Galpha(q) is essential for thrombin-induced CCL2 gene expression and protein release in murine lung fibroblasts. We further demonstrate that these effects are mediated via the cooperation between ERK1/2 and Rho kinase signaling pathways: a calcium-independent protein kinase C (PKC), c-Raf, and ERK1/2 pathway was found to mediate PAR(1)-induced CCL2 gene transcription, whereas a phospholipase C, calcium-dependent PKC, and Rho kinase pathway influences CCL2 protein release. We propose that targeting the interaction between PAR(1) and Galpha(q) may allow us to selectively interfere with PAR(1) proinflammatory and profibrotic signaling, while preserving the essential role of other PAR(1)-mediated cellular responses.

SUBMITTER: Deng X

PROVIDER: S-EPMC2397294 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Galphaq and cooperation between ERK1/2 and Rho kinase signaling pathways.

Deng Xiaoling X Mercer Paul F PF Scotton Chris J CJ Gilchrist Annette A Chambers Rachel C RC

Molecular biology of the cell 20080319 6

Uncontrolled activation of the coagulation cascade after tissue injury has been implicated in both inflammation and tissue fibrosis. Thrombin exerts pluripotent cellular effects via its high-affinity receptor, proteinase-activated receptor-1 (PAR(1)) and signaling via Galpha(i/o), Galpha(q), or Galpha(12/13). Activation of PAR(1) on fibroblasts, a key effector cell in fibrosis, results in the induction of several mediators, including the potent monocyte and fibrocyte chemoattractant CCL2. The ai ...[more]

PMID: 18353977

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Project description:Essentials Elimination of PDAC tumor cell PAR1 increased cytotoxic T cells and reduced tumor macrophages. PAR1KO PDAC cells are preferentially eliminated from growing tumors. Thrombin-PAR1 signaling in PDAC tumor cells drives an immunosuppressive gene signature. Csf2 and Ptgs2 are thrombin-PAR1 downstream immune suppressor genes in PDAC tumor cells. ABSTRACT: Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prothrombotic state and a lack of host antitumor immune responsiveness. Linking these two key features, we previously demonstrated that tumor-derived coagulation activity promotes immune evasion. Specifically, thrombin-protease-activated receptor-1 (PAR1) signaling in mouse PDAC cells drives tumor growth by evading cytotoxic CD8a+ cells. Methods Syngeneic mixed cell tumor growth, transcriptional analyses, and functional tests of immunosuppressive response genes were used to identify cellular and molecular immune evasion mechanisms mediated by thrombin-PAR-1 signaling in mouse PDAC tumor cells. Results Elimination of tumor cell PAR1 in syngeneic graft studies increased cytotoxic T lymphocyte (CTL) infiltration and decreased tumor-associated macrophages in the tumor microenvironment. Co-injection of PAR1-expressing and PAR1-knockout (PAR-1KO ) tumor cells into immunocompetent mice resulted in preferential elimination of PAR-1KO cells from developing tumors, suggesting that PAR1-dependent immune evasion is not reliant on CTL exclusion. Transcriptomics analyses revealed no PAR1-dependent changes in the expression of immune checkpoint proteins and no difference in major histocompatibility complex-I cell surface expression. Importantly, thrombin-PAR1 signaling in PDAC cells upregulated genes linked to immunosuppression, including Csf2 and Ptgs2. Functional analyses confirmed that both Csf2 and Ptgs2 are critical for PDAC syngeneic graft tumor growth and overexpression of each factor partially restored tumor growth of PAR1KO cells in immunocompetent mice. Conclusions Our results provide novel insight into the mechanisms of a previously unrecognized pathway coupling coagulation to PDAC immune evasion by identifying PAR1-dependent changes in the tumor microenvironment, a PAR1-driven immunosuppressive gene signature, and Csf2 and Ptgs2 as critical PAR1 downstream targets.

Project description:RationaleBMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown.ObjectiveWe aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models.Methods and resultsThe cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture-induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture-induced sepsis.ConclusionsActing as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.

Dataset Information

Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Galphaq and cooperation between ERK1/2 and Rho kinase signaling pathways.

Publications

Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Galphaq and cooperation between ERK1/2 and Rho kinase signaling pathways.

Similar Datasets

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