Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans.

The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4beta-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-beta-d-thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9.     

Thrombin induces NO release from cultured rat microglia via protein kinase C, mitogen-activated protein kinase, and NF-kappa B

Microglia, brain resident macrophages, become activated in brains injured due to trauma, ischemia, or neurodegenerative diseases. In this study, we found that thrombin treatment of microglia induced NO release/inducible nitric-oxide synthase expression, a prominent marker of activation. The effect of thrombin on NO release increased dose-dependently within the range of 5-20 units/ml. In immunoblot analyses, inducible nitric-oxide synthase expression was detected within 9 h after thrombin treatment. This effect of thrombin was significantly reduced by protein kinase C inhibitors, such as Go6976, bisindolylmaleimide, and Ro31-8220. Within 15 min, thrombin activated three subtypes of mitogen-activated protein kinases: extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase/stress-activated protein kinase. Inhibition of the extracellular signal-regulated kinase pathway and p38 reduced the NO release of thrombin-treated microglia. Thrombin also activated nuclear factor kappaB (NF-kappaB) within 5 min, and N-acetyl cysteine, an inhibitor of NF-kappaB, reduced NO release. However, thrombin receptor agonist peptide (an agonist of protease activated receptor-1 (PAR-1)), could not mimic the effect of thrombin, and cathepsin G, a PAR-1 inhibitor, did not reduce the effect of thrombin. These results suggest that thrombin can activate microglia via protein kinase C, mitogen-activated protein kinases, and NF-kappaB but that this occurs independently of PAR-1.     

Mechanisms of protease-activated receptor-4 actions in cardiomyocytes. Role of Src tyrosine kinase

Protease-activated receptor (PAR)-4 is a low affinity thrombin receptor with slow activation and desensitization kinetics relative to PAR-1. This study provides novel evidence that cardiomyocytes express functional PAR-4 whose signaling phenotype is distinct from PAR-1 in cardiomyocytes. AYPGKF, a modified PAR-4 agonist with increased potency at PAR-4, activates p38-mitogen-activated protein kinase but is a weak activator of phospholipase C, extracellular signal-regulated kinase, and cardiomyocyte hypertrophy; AYPGKF and thrombin, but not the PAR-1 agonist SFLLRN, activate Src. The observation that AYPGKF and thrombin activate Src in cardiomyocytes cultured from PAR-1(-/-) mice establishes that Src activation is via PAR-4 (and not PAR-1) in cardiomyocytes. Further studies implicate Src and epidermal growth factor receptor (EGFR) kinase activity in the PAR-4-dependent p38-mitogen-activated protein kinase signaling pathway. Thrombin phosphorylates EGFRs and ErbB2 via a PP1-sensitive pathway in PAR-1(-/-) cells that stably overexpress PAR-4; the Src-mediated pathway for EGFR/ErbB2 transactivation underlies the protracted phases of thrombin-dependent extracellular signal-regulated kinase activation in PAR-1(-/-) cells that overexpress PAR-4 and in cardiomyocytes. These studies identify a unique signaling phenotype for PAR-4 (relative to other cardiomyocyte G protein-coupled receptors) that is predicted to contribute to cardiac remodeling and influence the functional outcome at sites of cardiac inflammation.     

Exposure of cryptic domains in the alpha 1-chain of laminin-1 by elastase stimulates macrophages urokinase and matrix metalloproteinase-9 expression

Degradation of the extracellular matrix leads to the release of fragments, which elicit biological responses distinct from intact molecules. We have reported that alpha1:Ser(2091)-Arg(2108), a peptide derived from the alpha1-chain of laminin-1, triggers protein kinase C-dependent activation of MAPK(erk1/2), leading to the up-regulation of macrophage urokinase type plasminogen activator and matrix metalloproteinase (MMP)-9 expression. Since intact laminin-1 failed to trigger these events, we hypothesized that alpha1:Ser(2091)-Arg(2108) is cryptic or assumes a conformation not recognized by macrophages. Here we demonstrate that elastase cleavage of laminin-1 generates fragments, which stimulate proteinase expression by RAW264.7 macrophages and peritoneal macrophages. In contrast, fragments generated by MMP-2, MMP-7, or plasmin had no effect on macrophage proteinase expression. Elastase-generated laminin-1 fragments were fractionated by heparin-Sepharose chromatography. Heparin-binding fragments stimulated macrophages' proteinase expression severalfold greater than nonbinding fragments. The heparin binding fragments reacted with antibodies directed against regions of the alpha1-chain including alpha1:Ser(2091)-Arg(2108) and the globular domain. A peptide from the first loop of the globular domain (alpha1:Ser(2179)-Ser(2198)) triggered the phosphorylation of MAPK(erk1/2) and stimulated the expression of macrophage urokinase type plasminogen activator and MMP-9. Moreover, a heparin-binding fraction isolated from an aortic aneurysm contained fragments of alpha1-chain and stimulated macrophages' proteinase expression. Based on these data, we conclude that cryptic domains in the COOH-terminal portion of the alpha1-chain of laminin are exposed by proteolysis and stimulate macrophages' proteinase expression.     

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.     

Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1

Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin-induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up-regulated secretion of the chemokine growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) in primary rat astrocytes in a concentration-dependent manner. However, we found no increase of interleukin (IL)-6, IL-1beta and tumour necrosis factor-alpha secretion. Protease-activated receptor 1 (PAR-1)-induced GRO/CINC-1 release was mainly mediated by c-Jun N-terminal kinase (JNK) activation. Extracellular signal-regulated kinase 1/2 might be partially involved, but not p38 mitogen-activated protein kinase. Further studies demonstrated that PAR-1 activation, as well as application of recombinant GRO/CINC-1, protected astrocytes from C(2)-ceramide-induced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB-332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR-1. These results demonstrate for the first time that PAR-1 plays an important role in anti-apoptosis in the brain by regulating the release of chemokine GRO/CINC-1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.     

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.     

Mesotrypsin, a brain trypsin, activates selectively proteinase-activated receptor-1, but not proteinase-activated receptor-2, in rat astrocytes

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.     

Endothelial thrombomodulin induces Ca2+ signals and nitric oxide synthesis through epidermal growth factor receptor kinase and calmodulin kinase II

Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.     

Tissue-type plasminogen activator acts as a cytokine that triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression

Tissue-type plasminogen activator (tPA), a serine protease well known for generating plasmin, has been demonstrated to induce matrix metalloproteinase-9 (MMP-9) gene expression and protein secretion in renal interstitial fibroblasts. However, exactly how tPA transduces its signal into the nucleus to control gene expression is unknown. This study investigated the mechanism by which tPA induces MMP-9 gene expression. Both wild-type and non-enzymatic mutant tPA were found to induce MMP-9 expression in rat kidney interstitial fibroblasts (NRK-49F), indicating that the actions of tPA are independent of its proteolytic activity. tPA bound to the low density lipoprotein receptor-related protein-1 (LRP-1) in NRK-49F cells, and this binding was competitively abrogated by the LRP-1 antagonist, the receptor-associated protein. In mouse embryonic fibroblasts (PEA-13) lacking LRP-1, tPA failed to induce MMP-9 expression. Furthermore, tPA induced rapid tyrosine phosphorylation on the beta subunit of LRP-1, which was followed by the activation of Mek1 and its downstream Erk-1 and -2. Blockade of Erk-1/2 activation by the Mek1 inhibitor abolished MMP-9 induction by tPA in NRK-49F cells. Conversely, overexpression of constitutively activated Mek1 induced Erk-1/2 phosphorylation and MMP-9 expression. In mouse obstructed kidney, tPA, LRP-1, and MMP-9 were concomitantly induced in the renal interstitium. Collectively, these results suggest that besides its classical proteolytic activity, tPA acts as a cytokine that binds to the cell membrane receptor LRP-1, induces its tyrosine phosphorylation, and triggers intracellular signal transduction, thereby inducing specific gene expression in renal interstitial fibroblasts.     

Plasmin reduces fibronectin deposition by mesangial cells in a protease-activated receptor-1 independent manner

BackgroundProtease-activated receptor-1 (PAR-1) potentiates diabetic nephropathy (DN) as evident from reduced kidney injury in diabetic PAR-1 deficient mice. Although thrombin is the prototypical PAR-1 agonist, anticoagulant treatment does not limit DN in experimental animal models suggesting that thrombin is not the endogenous PAR-1 agonist driving DN.ObjectivesTo identify the endogenous PAR-1 agonist potentiating diabetes-induced nephropathy.MethodsUnbiased protease expression profiling in glomeruli from human kidneys with DN was performed using publically available microarray data. The identified prime candidate PAR-1 agonist was subsequently analysed for PAR-1-dependent induction of fibrosis in vitro.ResultsOf the 553 proteases expressed in the human genome, 247 qualified as potential PAR-1 agonists of which 71 were significantly expressed above background in diabetic glomeruli. The recently identified PAR-1 agonist plasmin(ogen), together with its physiological activator tissue plasminogen activator, were among the highest expressed proteases. Plasmin did however not induce mesangial proliferation and/or fibronectin deposition in vitro. In a PAR-1 independent manner, plasmin even reduced fibronectin deposition.ConclusionExpression profiling identified plasmin as potential endogenous PAR-1 agonist driving DN. Instead of inducing fibronectin expression, plasmin however reduced mesangial fibronectin deposition in vitro. Therefore we conclude that plasmin may not be the endogenous PAR-1 agonist potentiating DN.     

Early induction of matrix metalloproteinase-9 transduces signaling in human heart end stage failure

Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic-MMP-2 and -9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP-2 and -9 in ailing to failing myocardium. Activation of MMP-2 and -9 leads to induction of proteinase activated receptor-1 (PAR-1). We hypothesize that the early induction of MMP-9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase-1 (ERK-1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP-2 and -9 gelatinolytic activities was made by real-time quantitative zymography. Gel phosphorylation staining for PAR-1 showed a significant increase in ICM hearts. Western blot and RT-PCR analysis and in-situ labeling, showed significant increased expression of PAR-1, ERK-1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP-9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end-stage human heart failure.     

Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors.     

Thrombin up-regulates tissue factor pathway inhibitor-2 synthesis through a cyclooxygenase-2-dependent, epidermal growth factor receptor-independent mechanism

The serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) inhibits the tissue factor-factor VIIa complex and thereby impairs factor Xa and subsequently thrombin generation. Here we show that thrombin itself up-regulates TFPI-2 mRNA and protein expression in human liver myofibroblasts, a cell type shown to express high levels of TFPI-2 (Neaud, V., Hisaka, T., Monvoisin, A., Bedin, C., Balabaud, C., Foster, D. C., Desmoulière, A., Kisiel, W., and Rosenbaum, J. (2000) J. Biol. Chem. 275, 35565-35569). This effect required thrombin catalytic activity, as shown by its abolition with hirudin. Although the thrombin effect could be mimicked by agonists of both protease-activated receptor (PAR)-1 and PAR-4, it was largely blocked by a PAR-1 blocking antibody. Transactivation of the epidermal growth factor (EGF) receptor has been reported as a common event in thrombin signaling. However, thrombin did not detectably transactivate the EGF receptor in liver myofibroblasts, and blocking the EGF receptor did not affect TFPI-2 induction. On the other hand, thrombin increased the expression of cyclooxygenase-2 (COX-2) mRNA via a MAPK-dependent pathway, and a specific COX-2 inhibitor abolished the effect of thrombin on TFPI-2 expression. Thus, thrombin, through PAR-1 signaling, up-regulates the synthesis of TFPI-2 via a MAPK/COX-2-dependent pathway. The up-regulation of TFPI-2 expression by thrombin could in turn down-regulate thrombin generation and contribute to limit blood coagulation.     

Studies on the receptors mediating responses of osteoblasts to thrombin

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2'-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.     

Urokinase-type plasminogen activator stimulation of monocyte matrix metalloproteinase-1 production is mediated by plasmin-dependent signaling through annexin A2 and inhibited by inactive plasmin

Chronic inflammatory diseases are associated with connective tissue turnover that involves a series of proteases, which include the plasminogen activation system and the family of matrix metalloproteinases (MMPs). Urokinase-type plasminogen activator (uPA) and plasmin, in addition to their role in fibrinolysis and activation of pro-MMPs, have been shown to transduce intracellular signals through specific receptors. The potential for uPA and plasmin to also contribute to connective tissue turnover by directly regulating MMP production was examined in human monocytes. Both catalytically active high m.w. uPA, which binds to the uPAR, and low m.w. uPA, which does not, significantly enhanced MMP-1 synthesis by activated human monocytes. In contrast, the N-terminal fragment of uPA, which binds to uPAR, but lacks the catalytic site, failed to induce MMP-1 production, indicating that uPA-stimulated MMP-1 synthesis was plasmin dependent. Endogenous plasmin generated by the action of uPA or exogenous plasmin increased MMP-1 synthesis by signaling through annexin A2, as demonstrated by inhibition of MMP-1 production with Abs against annexin A2 and S100A10, a dimeric protein associated with annexin A2. Interaction of plasmin with annexin A2 resulted in the stimulation of ERK1/2 and p38 MAPK, cyclooxygenase-2, and PGE(2), leading to increased MMP-1 production. Furthermore, binding of inactive plasmin to annexin A2 inhibited plasmin induction of MMP-1, suggesting that inactive plasmin may be useful in suppressing inflammation.     

uPA Attenuated LPS-induced Inflammatory Osteoclastogenesis through the Plasmin/PAR-1/Ca2+/CaMKK/AMPK Axis

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis-caused bone destruction, results from an increase of bone-resorbing osteoclasts (OCs) induced by inflammation. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated that the effect of urokinase-type plasminogen activator (uPA) on inflammatory osteoclastogenesis induced by lipopolysaccharide (LPS), which is a potent stimulator of bone resorption in inflammatory diseases. We found that the uPA deficiency promoted inflammatory osteoclastogenesis and bone loss induced by LPS. We also showed that LPS induced the expression of uPA, and the uPA treatment attenuated the LPS-induced inflammatory osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells. Additionally, we showed that the uPA-attenuated inflammatory osteoclastgenesis is associated with the activation of plasmin/protease-activated receptor (PAR)-1 axis by uPA. Moreover, we examined the mechanism underlying the effect of uPA on inflammatory osteoclastogenesis, and found that uPA/plasmin/PAR-1 activated the adenosine monophosphate-activated protein kinase (AMPK) pathway through Ca2+/calmodulin dependent protein kinase kinase (CaMKK) activation, and attenuated inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 cells. These data suggest that uPA attenuated inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK axis. Our findings may provide a novel therapeutic approach to bone loss caused by inflammatory diseases.