Based on studies with the pCPB KO mice, we have obtained compelling data to support our general hypothesis that thrombin-activatable carboxypeptidase B (CPB or TAFIa) plays a broad anti-inflammatory role in vivo;the targets for CPB include bradykinin, C5a, the generation of plasmin and thrombin-cleaved osteopontin (OPN-Arg or OPN-R). We showed markedly elevated levels of OPN-R and its CPB-cleaved product, OPN-Leu (OPN-L), in synovial fluid samples from rheumatoid arthritis patients and these cleaved OPN forms affect neutrophils and fibroblast-like synoviocytes. Furthermore, we showed that prochemerin, a novel chemoattractant whose activity is dependent on C-terminal proteolytic cleavage by serine protease(s), can be activated by sequential cleavages by plasmin and CPB, thus establishing a new substrate for CPB and providing a novel molecular link between hemostasis/thrombosis and tissue inflammation and immunity.
Aim 1. To determine the biology of OPN-R and OPN-L in human glioblastoma (GBM). We demonstrated significantly elevated levels of OPN-R and OPN-L in CSF samples of patients with GBM. We will examine the distribution of OPN-FL, OPN-R, and OPN-L in GBM by immunohistology, and characterize cell signaling by OPN-R in comparison to OPN-FL and OPN-L in terms of cell adhesion, cell proliferation, apoptosis, protein phosphorylation, gene expression and cytokine production. We will test whether GBM cells, either at resting or stimulated state, will express and release OPN, resulting in cleaved OPN locally.
Aim 2. To determine the in vivo importance of thrombin cleavage of OPN using the thrombin non-cleavable OPN R153A knockin mice in inflammation, hemopoietic stem cell mobilization, aortic aneurysm, and cancer models. We have generated viable homozygous OPN R153A knockin (KI) mice and showed that recombinant OPN R153A is resistant to thrombin cleavage. Based on our preliminary data, we predicted that the homozygous OPN KI mice would be resistant to Con A-induced hepatitis. We will test whether the KI mice show enhanced hemopoietic stem cell mobilization and whether these animals are resistant to angiotensin II-induced abdominal aortic aneurysm formation in an ApoE null background. Since there is a close association between OPN and cancer, the KI mice will be tested in a chemical carcinogen (ENU)-induced cancer model to determine if abolition of OPN-R generation will alter the susceptibility to cancer development.
Aim 3. Biochemistry and biology of activation of prochemerin by serine/cysteine proteases and CPB. Active chemerin is present in GBM CSF samples (preliminary result) and RA synovial fluid. To determine the relevant forms of active chemerin in vivo, we have generated polyclonal antibodies specific for prochemerin and plasmin-cleaved and plasmin/CPB-double cleaved chemerin. These will be used to develop specific ELISAs to measure their levels in the clinical samples;we will determine the proteolytic cleavage efficiencies of prochemerin by different serine and cysteine proteases. Preliminary studies showed that GBM U87 cells express both chemerin and its receptors and that chemerin signals GBM U87 cells by STAT3 and S6 phosphorylation. We will examine whether GBM cells enhance plasmin generation and promote local active chemerin production. We will characterize chemerin signaling of U87 cells and test whether chemerin or post-culture media from GBM cells will recruit plasmacytoid dendritic cells. Our long-term goal is to understand the roles of thrombin and carboxypeptidase B and their cleavage products in the cross-talk between coagulation, tissue inflammation, and immunity, which has direct relevance to many pathological processes including malignancy, host defenses, and inflammatory diseases.
Thrombin is the main blood-clotting enzyme responsible for making a blood clot at the site of blood vessel injury. However, in addition to that, thrombin also activates many different types of cells and plays a key role in causing tissue inflammation. As part of a normal control mechanism, thrombin binds to a protein on the blood vessel surface called thrombomodulin and in that context, thrombin's function is "modulated'and will activate two other proteins, protein C and procarboxypeptidase B (pCPB). Activated protein C is now established as a protein that will dampen blood clotting, and exert a protective effect on blood vessels, thus serving as a feedback mechanism to down modulate thrombin's clotting and inflammatory functions. We and others have published data indicating that activated CPB may play a similar role in this process. The objective of this application is to study how CPB affects a number of specific inflammatory proteins and measure the products of this interaction to prove that these interactions actually occur in our body. We believe that this crosstalk between blood clotting, inflammation and immunity is important and highly relevant to our understanding of many diseases including cancer, inflammatory diseases, and abnormal blood clotting.
|Shao, Zhifei; Morser, John; Leung, Lawrence L K (2014) Thrombin cleavage of osteopontin disrupts a pro-chemotactic sequence for dendritic cells, which is compensated by the release of its pro-chemotactic C-terminal fragment. J Biol Chem 289:27146-58|
|Yamaguchi, Yasuto; Shao, Zhifei; Sharif, Shadi et al. (2013) Thrombin-cleaved fragments of osteopontin are overexpressed in malignant glial tumors and provide a molecular niche with survival advantage. J Biol Chem 288:3097-111|
|Yamaguchi, Yasuto; Du, Xiao-Yan; Zhao, Lei et al. (2011) Proteolytic cleavage of chemerin protein is necessary for activation to the active form, Chem157S, which functions as a signaling molecule in glioblastoma. J Biol Chem 286:39510-9|
|Zhao, Lei; Yamaguchi, Yasuto; Sharif, Shadi et al. (2011) Chemerin158K protein is the dominant chemerin isoform in synovial and cerebrospinal fluids but not in plasma. J Biol Chem 286:39520-7|
|Song, Jason J; Hwang, Inyong; Cho, Kyung H et al. (2011) Plasma carboxypeptidase B downregulates inflammatory responses in autoimmune arthritis. J Clin Invest 121:3517-27|
|Schultz, Geoffrey; Tedesco, Maureen M; Sho, Eiketsu et al. (2010) Enhanced abdominal aortic aneurysm formation in thrombin-activatable procarboxypeptidase B-deficient mice. Arterioscler Thromb Vasc Biol 30:1363-70|
|Mutch, N J; Myles, T; Leung, L L K et al. (2010) Polyphosphate binds with high affinity to exosite II of thrombin. J Thromb Haemost 8:548-55|
|Du, Xiao-Yan; Leung, Lawrence L K (2009) Proteolytic regulatory mechanism of chemerin bioactivity. Acta Biochim Biophys Sin (Shanghai) 41:973-9|
|Du, Xiao-Yan; Zabel, Brian A; Myles, Timothy et al. (2009) Regulation of chemerin bioactivity by plasma carboxypeptidase N, carboxypeptidase B (activated thrombin-activable fibrinolysis inhibitor), and platelets. J Biol Chem 284:751-8|
|Sharif, Shadi A; Du, Xiaoyan; Myles, Timothy et al. (2009) Thrombin-activatable carboxypeptidase B cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis. Arthritis Rheum 60:2902-12|
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