Xiao-Yan Du

Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets

Chemerin is a potent chemoattractant for cells expressing the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. The bioactivity of chemerin is post-translationally regulated; the attractant circulates in blood in a relatively inactive form (prochemerin) and is activated by carboxyl-terminal proteolytic cleavage. We discovered that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activable fibrinolysis inhibitor, TAFIa) enhanced the bioactivity of 10-mer chemerin peptide NH2-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH2-YFPGQFAFSKALPRS-COOH) or recombinant full-length prochemerin by plasmin and CPN/CPB substantially increased their chemotactic activities. Endogenous CPN present in circulating plasma enhanced the activity of plasmin-cleaved prochemerin. In addition, we discovered that platelets store chemerin protein and release it upon stimulation. Thus circulating CPN/CPB and platelets may potentially contribute to regulating the bioactivity of leukocyte chemoattractant chemerin, and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.

Thrombin-activatable carboxypeptidase B cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis.

OBJECTIVE Osteopontin (OPN) is a proinflammatory cytokine that plays an important role in the pathogenesis of rheumatoid arthritis (RA). OPN can be cleaved by thrombin, resulting in OPN-R and exposing the cryptic C-terminal alpha4beta1 and alpha9beta1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also called thrombin-activatable fibrinolysis inhibitor, removes the C-terminal arginine from OPN-R, generating OPN-L and abrogating its enhanced cell binding. We undertook this study to investigate the roles of OPN-R and OPN-L in synoviocyte adhesion, which contributes to the formation of invasive pannus, and in neutrophil survival, which affects inflammatory infiltrates in RA. METHODS Using specifically developed enzyme-linked immunosorbent assays, we tested the synovial fluid of patients with RA, osteoarthritis (OA), and psoriatic arthritis (PsA) to determine OPN-R, OPN-L, and full-length OPN (OPN-FL) levels. RESULTS Elevated levels of OPN-R and OPN-L were found in synovial fluid samples from RA patients, but not in samples from OA or PsA patients. Increased levels of OPN-R and OPN-L correlated with increased levels of multiple inflammatory cytokines, including tumor necrosis factor alpha and interleukin-6. Immunohistochemical analyses revealed robust expression of OPN-FL, but only minimal expression of OPN-R, in RA synovium, suggesting that cleaved OPN is released into synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an antiapoptotic effect on neutrophils. OPN-R augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to alpha4beta1, whereas OPN-L did not. CONCLUSION Thrombin activation of OPN (resulting in OPN-R) and its subsequent inactivation by thrombin-activatable CPB (generating OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA by regulating neutrophil viability and reducing synoviocyte adhesion.

Chemerin158K Protein Is the Dominant Chemerin Isoform in Synovial and Cerebrospinal Fluids but Not in Plasma

Background: Chemerin is a chemokine/adipokine whose activity depends on proteolytic processing. Results: Specific ELISAs demonstrate that in plasma the precursor is dominant, whereas in synovial fluid from arthritis patients and CSF from glioblastoma patients, chem158K dominates. Low levels of active chem157S were found. Conclusion: Chemerin proteolysis occurs during inflammation. Significance: This is the first report about levels of different chemerin isoforms in biological samples. Chemerin is a chemoattractant involved in immunity that may also function as an adipokine. Chemerin circulates as an inactive precursor (chem163S), and its activation requires proteolytic cleavages at its C terminus, involving proteases involved in coagulation, fibrinolysis, and inflammation. However, the key proteolytic steps in prochemerin activation in vivo remain to be established. Previously, we have shown that C-terminal cleavage of chem163S by plasmin to chem158K, followed by a carboxypeptidase cleavage, leads to the most active isoform, chem157S. To identify and quantify the in vivo chemerin isoforms in biological specimens, we developed specific ELISAs for chem163S, chem158K, and chem157S, using antibodies raised against peptides from the C terminus of the different chemerin isoforms. We found that the mean plasma concentrations of chem163S, chem158K, and chem157S were 40 ± 7.9, 8.1 ± 2.9, and 0.7 ± 0.8 ng/ml, respectively. The total level of cleaved and noncleaved chemerins in cerebrospinal fluids was ∼10% of plasma levels whereas it was elevated ∼2-fold in synovial fluids from patients with arthritis. On the other hand, the fraction of cleaved chemerins was much higher in synovial fluid and cerebrospinal fluid samples than in plasma (∼75%, 50%, and 18% respectively). Chem158K was the dominant chemerin isoform, and it was not generated by ex vivo processing, indicating that cleavage of prochemerin at position Lys-158, whether by plasmin or another serine protease, represents a major step in prochemerin activation in vivo. Our study provides the first direct evidence that chemerin undergoes extensive proteolytic processing in vivo, underlining the importance of measuring individual isoforms.

Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage

Background: Osteopontin (OPN) is highly expressed in glioblastoma (GBM) and possesses inflammatory activity modulated by proteolytic cleavage. Results: Cleaved OPN was increased in GBM and led to more adhesion of GBM cells. OPN conferred resistance to apoptosis in GBM cells. Conclusion: Increased osteopontin proteolysis increased GBM cell resistance to apoptosis. Significance: OPN cleavage links coagulation and inflammation providing a favorable niche for GBM development. Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPNRAA-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.

Proteolytic Cleavage of Chemerin Protein Is Necessary for Activation to the Active Form, Chem157S, Which Functions as a Signaling Molecule in Glioblastoma

Background: Chemerin (chem163S) is proteolytically cleaved to generate active isoforms. Results: Chem157S has the highest activity in stimulating calcium transients and chemotaxis, chem158K and chem163S have much lower activity, and chem155A is not an agonist. Chem157S stimulates signaling in glioblastoma cells. Conclusion: Chem163S proteolysis to chem157S is needed for its activity. Significance: Chemerin may play a role in glioblastoma. Chemerin is a chemoattractant involved in innate and adaptive immunity as well as an adipokine implicated in adipocyte differentiation. Chemerin circulates as an inactive precursor in blood whose bioactivity is closely regulated through proteolytic processing at its C terminus. We developed methodology for production of different recombinant chemerin isoforms (chem163S, chem157S, and chem155A) which allowed us to obtain large quantities of these proteins with purity of >95%. Chem158K was generated from chem163S by plasmin cleavage. Characterization by mass spectrometry and Edman degradation demonstrated that both the N and C termini were correct for each isoform. Ca2+ mobilization assays showed that the EC50 values for chem163S and chem158K were 54.2 ± 19.9 nm and 65.2 ± 13.2 nm, respectively, whereas chem157S had a ∼50-fold higher potency with an EC50 of 1.2 ± 0.7 nm. Chem155A had no agonist activity and weak antagonist activity, causing a 50% reduction of chem157S activity at a molar ratio of 100:1. Similar results were obtained in a chemotaxis assay. Because chem158K is the dominant form in cerebrospinal fluid from patients with glioblastoma (GBM), we examined the significance of chemerin in GBM biology. In silico analysis showed chemerin mRNA was significantly increased in tissue from grade III and IV gliomas. Furthermore, U-87 MG cells, a human GBM line, express the chemerin receptors, chemokine-like receptor 1 and chemokine receptor-like 2, and chem157S triggered Ca2+ flux. This study emphasized the necessity of appropriate C-terminal proteolytic processing to generate the likely physiologic form of active chemerin, chem157S, and suggested a possible role in malignant GBM.

Thrombospondin type I domain containing 7A (THSD7A) mediates endothelial cell migration and tube formation.

Angiogenesis is a highly organized process controlled by a series of molecular events. While much effort has been devoted to identifying angiogenic factors and their reciprocal receptors, far less information is available on the molecular mechanisms underlying directed endothelial cell migration. To search for novel proteins that participate in this process, we used the serial analysis of gene expression (SAGE) transcript profiling approach to identify genes that are selectively expressed in endothelial cells (ECs). Two EC SAGE libraries were constructed from human umbilical vein and artery ECs to enable data‐mining against other non‐ECs. A novel endothelial protein, Thrombospondin Type I Domain Containing 7A (THSD7A), with preferential expression in placenta vasculature and in human umbilical vein endothelial cells (HUVECs) was identified and targeted for further characterization. Overexpression of a THSD7A carboxyl‐terminal fragment in HUVECs inhibited cell migration and disrupted tube formation, while suppression of THSD7A expression enhanced HUVEC migration and tube formation. Immunohistological analysis revealed that THSD7A was expressed at the leading edge of migrating HUVECs, and it co‐localized with αVβ3 integrin and paxillin. This distribution was dispersed from focal adhesions after disruption of the actin cytoskeleton, suggesting the involvement of THSD7A in cytoskeletal organization. Our results show that THSD7A is a novel placenta endothelial protein that mediates EC migration and tube formation, and they highlight its potential as a new target for anti‐angiogenic therapy. J. Cell. Physiol. 222: 685–694, 2010.

Enhanced Abdominal Aortic Aneurysm Formation in Thrombin-Activatable Procarboxypeptidase B–Deficient Mice

Objective—To determine whether procarboxypeptidase B (pCPB)−/− mice are susceptible to accelerated abdominal aortic aneurysm (AAA) development secondary to unregulated OPN-mediated mural inflammation in the absence of CPB inhibition. Methods and Results—Thrombin/thrombomodulin cleaves thrombin-activatable pCPB or thrombin-activatable fibrinolysis inhibitor, activating CPB, which inhibits the generation of plasmin and inactivates proinflammatory mediators (complement C5a and thrombin-cleaved osteopontin [OPN]). Apolipoprotein E−/−OPN−/− mice are protected from experimental AAA formation. Murine AAAs were created via intra-aortic porcine pancreatic elastase (PPE) infusion. Increased mortality secondary to AAA rupture was observed in pCPB−/− mice at the standard PPE dose. At reduced doses of PPE, pCPB−/− mice developed larger AAAs than wild-type controls (1.01±0.27 versus 0.68±0.05 mm; P=0.02 [mean±SD]). C5−/− and OPN−/− mice were not protected against AAA development. Treatment with tranexamic acid inhibited plasmin generation and abrogated enhanced AAA progression in pCPB−/− mice. Conclusion—This study establishes the role of CPB in experimental AAA disease, indicating that CPB has a broad anti-inflammatory role in vivo. Enhanced AAA formation in the PPE model is the result of increased plasmin generation, not unregulated C5a- or OPN-mediated mural inflammation.