Proteolytic regulation of the urokinase receptor/CD87 on monocytic cells by neutrophil elastase and cathepsin G

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.     

Effect of DNase on the activity of neutrophil elastase, cathepsin G and proteinase 3 in the presence of DNA

It has been shown previously that DNA binds and inhibits neutrophil elastase (NE). Here we demonstrate that DNA has a better affinity for neutrophil cathepsin G (cat G) than for NE and is a better inhibitor of cat G than of NE. DNase-generated <0.5 kb DNA fragments inhibit NE and cat G as potently as full length DNA. This rationalises our observation that administration of DNase to cystic fibrosis patients does not enhance the NE and cat G activity of their lung secretions. Neutrophil proteinase 3 is not inhibited by DNA and might thus be the most harmful proteinase in inflammatory lung diseases.     

Phosphoinositide 3-kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing alpha(IIb)beta(3) integrin activation

Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.     

Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions

Polymorphonuclear neutrophils form a primary line of defense against bacterial infections using complementary oxidative and non-oxidative pathways to destroy phagocytized pathogens. The three serine proteases elastase, proteinase 3 and cathepsin G, are major components of the neutrophil primary granules that participate in the non-oxidative pathway of intracellular pathogen destruction. Neutrophil activation and degranulation results in the release of these proteases into the extracellular medium as proteolytically active enzymes, part of them remaining exposed at the cell surface. Extracellular neutrophil serine proteases also help kill bacteria and are involved in the degradation of extracellular matrix components during acute and chronic inflammation. But they are also important as specific regulators of the immune response, controlling cellular signaling through the processing of chemokines, modulating the cytokine network, and activating specific cell surface receptors. Neutrophil serine proteases are also involved in the pathogenicity of a variety of human diseases. This review focuses on the structural and functional properties of these proteases that may explain their specific biological roles, and facilitate their use as molecular targets for new therapeutic strategies.     

Enhanced release of oxygen metabolites by monocyte-derived macrophages exposed to proteolytic enzymes: activity of neutrophil elastase and cathepsin G

Macrophages at sites of inflammation are exposed to proteolytic enzymes derived from neutrophils, platelets, clotting factors, complement, and damaged tissues. To investigate the possible effect of proteases on the plasma membrane-mediated oxidative metabolic response of macrophages in inflammatory sites, cultured human monocyte-derived macrophages were treated in vitro with proteolytic enzymes and were then assayed for their ability to release superoxide anion (O2-) and hydrogen peroxide (H2O2) in response to stimuli. Macrophages pretreated for 1 to 20 min with trypsin, chymotrypsin, pronase, or papain, 0.1 to 200 micrograms/ml, released up to 3.5-times more O2- and H2O2 than did control (untreated) cells. This enhanced production of oxygen metabolites was observed by using either phorbol myristate acetate or opsonized zymosan as the stimulus. Macrophages were also "primed" for enhanced O2- release (2.3-fold) by pretreatment with a subfraction of granules extracted from human neutrophils. This subfraction contained primarily elastase and cathepsin G. Similar enhancement was observed with 60 ng/ml or purified human neutrophil cathepsin G (2.2-fold) and with 20 micrograms/ml of purified neutrophil elastase (3.3-fold). Priming by these neutrophil proteases could be blocked by specific inhibitors of their proteolytic activity. These results suggest that macrophages involved in an inflammatory response might be rapidly primed by proteases released from degranulating neutrophils. Primed macrophages could mount a more effective oxidative metabolic response to microorganisms or tumor cells, but might also cause greater tissue damage.     

Inhibition of serine proteinases plasmin,trypsin, subtilisin A,cathepsin G,and elastase by LEKTI: a kinetic analysis

The human LEKTI gene encodes a putative 15-domain serine proteinase inhibitor and has been linked to the inherited disorder known as Netherton syndrome. In this study, human recombinant LEKTI (rLEKTI) was purified using a baculovirus/insect cell expression system, and the inhibitory profile of the full-length rLEKTI protein was examined. Expression of LEKTI in Sf9 cells showed the presence of disulfide bonds, suggesting the maintenance of the tertiary protein structure. rLEKTI inhibited the serine proteinases plasmin, subtilisin A, cathepsin G, human neutrophil elastase, and trypsin, but not chymotrypsin. Moreover, rLEKTI did not inhibit the cysteine proteinase papain or cathepsin K, L, or S. Further, rLEKTI inhibitory activity was inactivated by treatment with 20 mM DTT, suggesting that disulfide bonds are important to LEKTI function. The inhibition of plasmin, subtilisin A, cathepsin G, elastase, and trypsin by rLEKTI occurred through a noncompetitive-type mechanism, with inhibitory constants (K(i)) of 27 +/- 5, 49 +/- 3, 67 +/- 6, 317 +/-36, and 849 +/- 55 nM, respectively. Thus, LEKTI is likely to be a major physiological inhibitor of multiple serine proteinases.     

Isoxazol-5(2H)-one: a new scaffold for potent human neutrophil elastase (HNE) inhibitors

Human neutrophil elastase (HNE) is an important target for the development of novel and selective inhibitors to treat inflammatory diseases, especially pulmonary pathologies. Here, we report the synthesis, structure–activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with an isoxazol-5(2H)-one scaffold. The most potent compound (2o) had a good balance between HNE inhibitory activity (IC₅₀ value =20?nM) and chemical stability in aqueous buffer (t_1/2=8.9?h). Analysis of reaction kinetics revealed that the most potent isoxazolone derivatives were reversible competitive inhibitors of HNE. Furthermore, since compounds 2o and 2s contain two carbonyl groups (2-N-CO and 5-CO) as possible points of attack for Ser195, the amino acid of the active site responsible for the nucleophilic attack, docking studies allowed us to clarify the different roles played by these groups.     

Synthetic inhibitors of human granulocyte elastase, Part 4. Inhibition of human granulocyte elastase and cathepsin G by non-steroidal anti-inflammatory drugs (NSAID)s

A series of non-steroidal anti-inflammatory drugs (NSAIDs) and some of their metabolites were evaluated for their ability to inhibit the proteolytic activity of human granulocyte elastase (HGE) and cathepsin G (HGC-G). The enzyme activity was monitored using specific synthetic chromogenic substrates. The results obtained indicated that phenylbutazone, sulindac, piroxicam and gold sodium thiomalate significantly inhibited HGE (Ki less than 0.5 mM), while only sulindac, diflunisal and gold sodium thiomalate were effective inhibitors of HGC-G (Ki less than 0.4 mM). Studies on metabolites of some of the NSAID tested were found to be superior inhibitors of both HGE and HGC-G than the parent molecules. Moreover, of the 18 compounds examined, the major metabolite of sulindac, sulindac sulphide was the most potent inhibitor of HGE (Ki = 0.01 mM) and HGC-G (Ki = 0.15 mM).     

Suppression of KSHV-induced angiopoietin-2 inhibits angiogenesis,infiltration of inflammatory cells,and tumor growth

Kaposi's sarcoma (KS) is a highly angiogenic and inflammatory neoplasia. The angiogenic and inflammatory cytokine angiopoietin-2 (Ang-2) is strongly expressed in KS due to Kaposi's sarcoma-associated herpesvirus (KSHV) infection. In the present study, we determined how Ang-2 contributes to development of KS by using telomerase-immortalized human umbilical vein endothelial cells (TIVE) as a model, which become malignantly transformed and express increased levels of Ang-2 following KSHV infection. Ang-2 released from TIVE-KSHV cells induces tyrosine phosphorylation of Tie-2 receptor from both human and mouse endothelial cells and promotes angiogenesis in nude mice. Functional inhibition or expressional “knock-down” of Ang-2 in these cells blocks angiogenesis and inhibits tumor growth. Ang-2 suppression also reduces the numbers of infiltrating monocytes/macrophages in tumors. In transwell-based cell migration assays, Ang-2 indeed enhances migration of human monocytes in a dose-dependent manner. These results underscore a pivotal role of KSHV-induced Ang-2 in KS tumor development by promoting both angiogenesis and inflammation. Our data also suggest that selective drug targeting of Ang-2 may be used for treatment of KS.     

Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/stromelysin-1 cascade enhances tumor cell invasion

Matrix metalloproteinase-9 (MMP-9) may play a critical catalytic role in tissue remodeling in vivo, but it is secreted by cells as a stable, inactive zymogen, pro-MMP-9, and requires activation for catalytic function. A number of proteolytic enzymes activate pro-MMP-9 in vitro, but the natural activator(s) of MMP-9 is unknown. To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells (MDA-MB-231 breast carcinoma cells) that were induced to produce MMP-9 over a 200-fold concentration range (0.03-8.1 nM). The levels of tissue inhibitors of metalloproteinase (TIMPs) in the induced cultures remain relatively constant at 1-4 nM. Quantitation of the zymogen/active enzyme status of MMP-9 in the MDA-MB-231 cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether pro-MMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but through an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous urokinase-type plasminogen activator, is not an efficient activator of pro-MMP-9, neither the secreted pro-MMP-9 nor the very low levels of pro-MMP-9 associated with intact cells. Although plasmin can proteolytically process pro-MMP-9, this limited action does not yield an enzymatically active MMP-9, nor does it cause the MMP-9 to be more susceptible to activation. Plasmin, however, is very efficient at generating active MMP-3 (stromelysin-1) from exogenously added pro-MMP-3. The activated MMP-3 becomes a potent activator of the 92-kDa pro-MMP-9, yielding an 82-kDa species that is enzymatically active in solution and represents up to 50-75% conversion of the zymogen. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to both degrade extracellular matrix and transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodelling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9 blocking monoclonal antibody.     

Downregulation of uPA, uPAR and MMP-9 using small, interfering, hairpin RNA (siRNA) inhibits glioma cell invasion, angiogenesis and tumor growth

The diffuse, extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modification of the proteolysis of extracellular matrix components. Our previous results clearly demonstrate that uPA, uPAR and MMP-9 concentrations increase significantly during tumor progression and that tumor growth can be inhibited with antisense stable clones of these molecules. Because antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPA, uPAR and MMP-9 in this study. We examined a cytomegalovirus (CMV) promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to inhibit uPA, uPAR and MMP-9 gene expression with a single construct. uPAR protein levels and enzymatic activity of uPA and MMP-9 were found to significantly decrease in cells transfected with a plasmid expressing hairpin siRNA for uPAR, uPA and MMP-9. pU(2)M-transfected SNB19 cells significantly decreased uPA, uPAR and MMP-9 expression compared to mock and EV/SV-transfected cells, determined by immunohistochemical analysis. Furthermore, the effect of the single constructs for these molecules was a specific inhibition of their respective protein levels, as demonstrated by immunohistochemical analysis. After transfection with a plasmid vector expressing dsRNA for uPA, uPAR and MMP-9, glioma-cell invasion was retarded compared with mock and EV/SV-treated groups, demonstrated by Matrigel-invasion assay and spheroid-invasion assay. Downregulation of uPA, uPAR and MMP-9 using RNAi inhibited angiogenesis in an in vitro (co-culture) model. Direct intratumoral injections of plasmid DNA expressing hpRNA for uPA, uPAR and MMP-9 significantly regressed pre-established intracranial tumors in nude mice. In addition, cells treated with RNAi for uPAR, uPA and MMP-9 showed reduced pERK levels compared with parental and EV/SV-treated SNB19 cells. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating gliomas.     

Thromboxane A(2) regulation of endothelial cell migration, angiogenesis, and tumor metastasis

Prostaglandin endoperoxide H synthases and their arachidonate products have been implicated in modulating angiogenesis during tumor growth and chronic inflammation. Here we report the involvement of thromboxane A(2), a downstream metabolite of prostaglandin H synthase, in angiogenesis. A TXA(2) mimetic, U46619, stimulated endothelial cell migration. Angiogenic basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) increased TXA(2) synthesis in endothelial cells three- to fivefold. Inhibition of TXA(2) synthesis with furegrelate or CI reduced HUVEC migration stimulated by VEGF or bFGF. A TXA(2) receptor antagonist, SQ29,548, inhibited VEGF- or bFGF-stimulated endothelial cell migration. In vivo, CI inhibited bFGF-induced angiogenesis. Finally, development of lung metastasis in C57Bl/6J mice intravenously injected with Lewis lung carcinoma or B16a cells was significantly inhibited by thromboxane synthase inhibitors, CI or furegrelate sodium. Our data demonstrate the involvement of TXA(2) in angiogenesis and development of tumor metastasis.     

A novel locust (Schistocerca gregaria) serine protease inhibitor with a high affinity for neutrophil elastase

We have purified to homogeneity two forms of a new serine protease inhibitor specific for elastase/chymotrypsin from the ovary gland of the desert locust Schistocerca gregaria. This protein, greglin, has 83 amino acid residues and bears putative phosphorylation sites. Amino acid sequence alignments revealed no homology with pacifastin insect inhibitors and only a distant relationship with Kazal-type inhibitors. This was confirmed by computer-based structural studies. The most closely related homologue is a putative gene product from Ciona intestinalis with which it shares 38% sequence homology. Greglin is a fast-acting and tight binding inhibitor of human neutrophil elastase (k(ass)=1.2x10(7) M(-1) x s(-1), K(i)=3.6 nM) and subtilisin. It also binds neutrophil cathepsin G, pancreatic elastase and chymotrypsin with a lower affinity (26 nM< or =K(i)< or =153 nM), but does not inhibit neutrophil protease 3 or pancreatic trypsin. The capacity of greglin to inhibit neutrophil elastase was not significantly affected by exposure to acetonitrile, high temperature (90 degrees C), low or high pH (2.5-11.0), N-chlorosuccinimide-mediated oxidation or the proteolytic enzymes trypsin, papain and pseudolysin from Pseudomonas aeruginosa. Greglin efficiently inhibits the neutrophil elastase activity of sputum supernatants from cystic fibrosis patients. Its biological function in the locust ovary gland is currently unknown, but its physicochemical properties suggest that it can be used as a template to design a new generation of highly resistant elastase inhibitors for treating inflammatory diseases.     

Modulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) by interferon-γ in a human salivary gland cell line

Gelatinases have been shown to be regulated by many cytokines and growth factors, and have been implicated in the pathogenesis of certain autoimmune diseases via tissue destruction. High levels of several cytokines, including IFN-γ and TNF-α, have been demonstrated in the salivary gland microenvironment of patients with Sjo¨︁gren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of this disease is not well understood. We hypothesized that IFN-γ with or without (±) TNF-α could be playing a role in the pathogenesis of SS via the regulation of matrix metalloproteinase (MMP) levels. This study examined the role of IFN-γ and (+) TNF-α in the regulation of the matrix metalloproteinases, MMP-2 (72 kD gelatinase A) and MMP-9 (92 kD gelatinase B). A human salivary gland cell line (HSG) has been used as a possible in vitro model to study the role of IFN-γ + TNF-α in the pathogenesis of SS. The HSG cell line, in the presence of IFN ± TNF-α, displays increased MMP-2 and MMP-9 gelatinolytic activity, protein and RNA levels. The increase in MMP activity was partially blocked with an antibody against the IFN-γ receptor, and this was associated with a complete inhibition of the previously described IFN-γ ± TNF-α antiproliferative effect. However, incubation of IFN-γ treated HSG cells with the synthetic MMP inhibitor BB94 did not alleviate this antiproliferative effect. In addition, we demonstrate that there are very high levels of MMP-9 in the saliva of patients with SS when compared to healthy control subjects. These data suggest that cytokines could be regulating MMP production by salivary epithelial cells and thus indicate a potential role for these cells in the pathogenesis of SS. J. Cell. Physiol. 171:117–124, 1997. © 1997 Wiley-Liss, Inc.     

Promoter polymorphism MMP-1 (-1607 2G/1G) and MMP-3 (-1612 5A/6A) in development of HAND and modulation of pathogenesis of HAND

The pathogenesis of HIV-associated neurocognitive disorder (HAND) is modulated by host genetic susceptibility factors such as Matrix metalloproteinases (MMPs). Promoter polymorphism of MMP-1 and MMP-3 may modify the expression of the gene. Hence, we evaluated the association of MMP-1-16072G/1G and MMP-3-1612 5A/6A polymorphisms with development of HAND and the modulation of pathogenesis of HAND. We enrolled a total of 180 individuals, 50 HIV-infected individuals with HAND, 130 without HAND, and 150 healthy controls. Polymorphism of MMP-1 and MMP-3 were genotyped by PCR-RFLP. MMP-1-1607 2G1G, -16071G/2G-1G/1G genotypes and -1607 1G allele were associated with the development of HAND (OR = 1.64, P = 0.05; OR = 1.45, P = 0.04; OR = 1.69, P = 0.05). MMP-1-16071G1G, MMP-3-16125A5A genotypes increased the risk for the development of HAND (OR = 1.78, P = 0.25; OR = 2.39, P = 0.13). MMP-3-1612 5A5A, -1612 6A/5A-5A/5A genotypes and -1612 5A allele were associated with the reduced risk of HAND (OR = 0.40, P = 0.05; OR = 0.53, P = 0.04; OR = 0.40, P = 0.01). Haplotype 5A1G increased the risk of development of HAND (OR = 1.93, P = 0.05). As observed in advanced HIV disease stage, MMP-1-1607 1G1G genotype enhance the risk for advancement of HIV disease (OR = 1.69, P = 0.89). MMP-3-1612 6A5A genotype showed higher risk for development of HAND in alcohol users (0R = 1.65, P = 0.44). MMP-1 genotype may have an influence on development of HAND whereas MMP3-1612 5A5A genotype may reduce risk for pathogenesis of HAND.     

Activation of interstitial collagenase, MMP-1, by Staphylococcus aureus cells having surface-bound plasmin: a novel role of plasminogen receptors of bacteria

Plasmin, the enzymatically active form of plasminogen, can activate several matrix metalloproteinases (MMPs). In this study, we investigated the activation of MMP-1, one of the major interstitial collagenases, by plasmin which was generated on the surface of Staphylococcus aureus cells. Plasmin bound to plasminogen receptors on S. aureus degraded the major (125)I-labeled 55-kDa proMMP-1 into the 42-kDa form corresponding to the size of active MMP-1. MMP-1 formed by S. aureus-bound plasmin was also enzymatically active as judged by digestion of the synthetic collagenase substrate, DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH(2). The finding that, in MMP-1 molecules generated either by soluble plasmin or by S. aureus-bound plasmin, the amino-terminal amino acid sequences were identical indicated that the activation mechanisms of the two plasmin forms do not differ from each other. The present observations emphasise and broaden the physiological importance of bacterial plasminogen receptors. In addition to direct proteolytic effects on components of the extracellular matrix, receptor-bound plasmin is also capable of initiating an MMP-1-dependent matrix-degrading enzymatic cascade.     

Retracted: miR-942 promotes tumor migration,invasion, and angiogenesis by regulating EMT via BARX2 in non-small-cell lung cancer

Epithelial–mesenchymal transition (EMT) has an important function in cancer. Recently, microRNAs have been reported to be involved in EMT by regulating target genes. miR-942 is considered a novel oncogene in esophageal squamous cell carcinoma. However, its role in non-small-cell lung cancer (NSCLC) has not been investigated. In this study, the expression of miR-942 in NSCLC patients tumor and paired adjacent tissues were assessed by quantitative real-time polymerase chain reaction and in situ hybridization. Transwell, wound healing, tube formation, and tail vein xenograft assays were conducted to assess miR-942′s function in NSCLC. Potential miR-942 targets were confirmed using dual-luciferase reporter assays, immunohistochemistry, immunoblot, and rescue experiments. The results showed miR-942 is relatively highly expressed in human NSCLC tissues and cells. In vitro assays demonstrated that overexpression of miR-942 promoted cell migration, invasion, and angiogenesis. Tail vein xenograft assays suggested that miR-942 contributed to NSCLC metastasis in vivo. Three bioinformatics software was searched, and BARX2 was predicted as a downstream target of miR-942. Direct interaction between them was validated by dual-luciferase assays. Rescue experiments further confirmed that BARX2 overexpression could reverse functional changes caused by miR-942. Moreover, miR-942 increased EMT-associated proteins N-cadherin and vimentin by inhibiting BARX2, while E-cadherin expression is reduced. In summary, this study reveals that miR-942 induces EMT-related metastasis by directly targeting BARX2, which may provide a potential therapeutic strategy for NSCLC.