Catechins prevent vascular smooth muscle cell invasion by inhibiting MT1-MMP activity and MMP-2 expression

OBJECTIVE: Regular consumption of green tea is associated with a reduced risk of mortality due to coronary diseases and cancer. The present study examined whether a green tea extract (GTE) inhibits activation of matrix metalloproteinase-2 (MMP-2), a major collagenase involved in vascular remodeling of atherosclerotic plaques, in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: The expression of MMP-2 was assessed by Northern and Western blot analyses in human aortic VSMCs. MMP-2 activity was evaluated by zymography, membrane-type1-MMP (MT1-MMP, MMP-14) activity by an enzymatic assay, and cell invasion by a modified Boyden chamber assay. The thrombin-induced activation of secreted MMP-2 was abolished by GTE and the green tea polyphenols (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG). GTE reduced the expression of MMP-2 mRNA and protein. GTE, EGCG and ECG directly inhibited cell-associated MT1-MMP activity, the physiological activator of MMP-2, in a reversible manner. Thrombin-stimulated VSMCs invasion was abolished by EGCG and ECG, and reduced by GTE. CONCLUSIONS: GTE inhibits thrombin-induced VSMCs invasion most likely by preventing MMP-2 expression and its activation by a direct inhibition of MT1-MMP. The ability of green tea to prevent cell invasion and matrix degradation might contribute to its protective effect on atherosclerosis and cancer.     

Apoptosis of human hepatic myofibroblasts promotes activation of matrix metalloproteinase-2

Liver fibrosis is potentially reversible after removal of the injurious agent. Fibrosis resolution is characterized by apoptosis of hepatic myofibroblasts and degradation of extracellular matrix components. Matrix metalloproteinase-2 (MMP-2) is involved in matrix remodeling. In the liver, it is synthesized by myofibroblasts, secreted as a proenzyme, and activated by membrane type-MMPs (MT-MMP) such as MT1-MMP. The goal of this work was to determine whether apoptosis induction in human hepatic myofibroblasts modulates the gene expression of MMP-2 and/or its activation by MT1-MMP. Induction of apoptosis by cytochalasin D or C(2)-ceramide did not modulate MMP-2 mRNA expression. In contrast, apoptosis was associated with marked activation of pro-MMP-2, as shown by gelatin zymography, which revealed the presence of the 59-kd active form, whereas untreated cells only expressed the 66-kd proform. SB-203580, a specific inhibitor of p38 (MAPK), selectively abrogated both C(2)-ceramide-induced apoptosis and pro-MMP-2 activation. Apoptosis-induced pro-MMP-2 activation was inhibited by the tissue inhibitors of metalloproteinases (TIMP)-2 but not by TIMP-1, implying involvement of an MT-MMP-mediated process. Induction of apoptosis by cytochalasin D and C(2)-ceramide upregulated MT1-MMP protein expression and MT1-MMP mRNA expression. In conclusion, apoptosis of hepatic myofibroblasts induces pro-MMP-2 activation through increased MT1-MMP expression. HEPATOLOGY 2002;36:615-622.)     

Regulation of MT1-MMP and MMP-2 by leptin in cardiac fibroblasts involves Rho/ROCK-dependent actin cytoskeletal reorganization and leads to enhanced cell migration

Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells. Coimmunoprecipitation analysis showed that leptin also induced the formation of a cluster of differentiation 44/MT1-MMP complex. Qualitative analysis using rhodamine-conjugated phalloidin immunofluorescence indicated that leptin stimulated actin cytoskeletal reorganization and enhanced stress fiber formation. Hence, we analyzed activation of Ras homolog gene family (Rho), member A GTPase activity and found a rapid increase in response to leptin that corresponded with increased phosphorylation of cofilin. Quantitative analysis of cytoskeleton reorganization upon separation of globular and filamentous actin by differential centrifugation confirmed the significant increase in filamentous to globular actin ratio in response to leptin, which was prevented by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated coiled-coil-forming protein kinase (ROCK) (Y-27632). Inhibition of Rho or ROCK also attenuated leptin-stimulated increases in cell surface MT1-MMP content. Pro-MMP-2 is a known MT1-MMP substrate, and we observed that enhanced cell surface MT1-MMP in response to leptin resulted in enhanced extracellular activation of pro-MMP-2 measured by gelatin zymography, which was again attenuated by inhibition of Rho or ROCK. Using wound scratch assays, we observed enhanced cell migration, but not proliferation, measured by 5-bromo2'-deoxy-uridine incorporation, in response to leptin, again via a Rho-dependent signaling mechanism. Our results suggest that leptin regulates myocardial matrix remodeling by regulating the cell surface localization of MT1-MMP in adult cardiac fibroblasts via Rho/ROCK-dependent actin cytoskeleton reorganization. Subsequent pro-MMP-2 activation then contributes to stimulation of cell migration.     

Induction of membrane-type matrix metalloproteinase-1 stimulates angiogenic activities of bovine aortic endothelial cells

Matrix metalloproteinases (MMPs) have been reported to play critical roles in endothelial cell migration and matrix remodeling during the angiogenic process. Among these MMPs, membrane-type MMP-1 (MT1-MMP) is an important molecule that can trigger the invasion of tumor cells by activating MMP-2 on their plasma membrane. However, the precise involvement of MT1-MMP in the angiogenic process has not been determined. To investigate the roles of the MT1-MMP by the matrix remodeling of endothelial cells, MT1-MMP expression vector was transfected into bovine aortic endothelial cells (BAECs). Increased expression of MT1-MMP in BAECs enhanced the activation of MMP-2, invasion and migration of BAECs. Moreover, the capacity of tube formation was increased in MT1-MMP transfectants. However, cotransfection with antisense MT1-MMP expression vector abolished the effects of MT1-MMP overexpression. These observations indicate that MT1-MMP is involved in the angiogenic process of endothelial cells in vitro. This revised version was published online in June 2006 with corrections to the Cover Date.     

成纤维细胞与肺癌细胞相互作用对膜型基质金属蛋白酶-1及基质金属蛋白酶-2表达的影响

目的 研究胚肺成纤维细胞对肺癌H460细胞膜型基质金属蛋白酶-1(MTl-MMP)、基质金属蛋白酶-2(MMP-2)表达的影响.方法 采用Western blot方法检测各组MT1-MMP的表达.取其上清,采用酶联免疫吸附法检测各组细胞培养液中活性MMP-2的浓度.结果 H460细胞、胚肺成纤维细胞单独培养时均有MT1-MMP表达,但混合培养后MT1-MMP表达增强(P＜0.05).H460细胞、胚肺成纤维细胞单独培养时MMP-2均有分泌,混合培养MMP-2分泌增强(P＜0.05).结论 胚肺成纤维细胞和肺癌H460细胞相互作用能通过上调MT1-MMP、MMP-2的表达从而促进肺癌的侵袭和转移,这可能为肺癌侵袭转移的一个重要机制.     

Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-beta1 involves furin-convertase

OBJECTIVE: Heart failure is characterized by an imbalance of matrix synthesis/turnover, finally resulting in fibrosis. Cardiac myocytes and fibroblasts play a pivotal role in the remodeling process. Cardiac remodeling involves the expression of TGF-beta1 and matrix metalloproteinases (MMPs) in cardiac fibroblasts (CFBs). Furin, a subtilisin/kexin-like proprotein convertase (PC), activates TGF-beta1 and membrane-bound MT1-MMP, which facilitates pro-gelatinase A (MMP-2) activation. Even though several reports identified TGF-beta1 as a pro-fibrotic cytokine in the heart, it increases MMP-activity and cell migration/invasion in several cell types. The present study was done to investigate the contribution of TGF-beta1 and furin to CFBs MMP-activity and motility. METHODS AND RESULTS: Stimulation of CFBs from adult Sprague-Dawley rats with TGF-beta1 (20 ng/ml) induced furin, but had no effect on the closely related PC5. Inhibition of furin inhibited angiotensin II-induced TGF-beta1 activation, indicating that TGF-beta1 amplifies its activating convertase in CFBs. Pretreatment of CFBs with TGF-beta1 (20 ng/ml, 24 h) increased their migration by about two-fold (p<0.05), which was accompanied by an enhanced expression and activity of MT1-MMP and MMP-2. Brefeldin A (BFA), a Golgi-disturbing agent, inhibited MT1-MMP activation, indicating that it occurs in the trans-Golgi network (TGN), where furin is concentrated and colocalized with MT1-MMP. Inhibition of furin significantly inhibited TGF-beta1-induced MT1-MMP/MMP-2 activation. Furthermore, inhibition of furin attenuated TGF-beta1-enhanced migration on gelatin-coated membranes (p<0.05). This was comparable to the effects of the MMP-inhibitor GM6001, pointing out that MMPs are major mediators of TGF-beta1-enhanced CFB motility. CONCLUSION: We demonstrate that TGF-beta1 induces MMP-activity in CFBs, thereby facilitating CFBs motility. Furthermore, TGF-beta1 amplifies its activating convertase furin, which is also required for MT1-MMP/MMP-2 activation in CFBs. Thus, furin is central for TGF-beta1 and MT1-MMP activation and might be a novel target in cardiac remodeling.     

Chronic hypoxia inhibits MMP-2 activation and cellular invasion in human cardiac myofibroblasts

Cardiac myofibroblasts are pivotal to adaptive remodelling after myocardial infarction (MI). These normally quiescent cells invade and proliferate as a wound healing response, facilitated by activation of matrix metalloproteinases, particularly MMP-2. Following MI these reparative events occur under chronically hypoxic conditions yet the mechanisms by which hypoxia might modulate MMP-2 activation and cardiac myofibroblast invasion have not been investigated. Human cardiac myofibroblasts cultured in collagen-supplemented medium were exposed to normoxia (20% O₂) or hypoxia (1% O₂) for up to 48 h. Secreted levels of total and active MMP-2 were quantified using gelatin zymography, TIMP-2 and membrane-associated MT1-MMP were quantified with ELISA, whole cell MT1-MMP by immunoblotting and immunocytochemistry and MT1-MMP mRNA with real-time RT-PCR. Cellular invasion was assessed in modified Boyden chambers and migration by scratch wound assay.In the human cardiac myofibroblast, MT1-MMP was central to MMP-2 activation and activated MMP-2 necessary for invasion, confirmed by gene silencing. MMP-2 activation was substantially attenuated by hypoxia (P < 0.001), paralleled by inhibition of myofibroblast invasion (P < 0.05). In contrast, migration was independent of either MT1-MMP or MMP-2. Reduced membrane expression of MT1-MMP (P < 0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2. In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI. Regulation of these MMPs remains an attractive target for therapeutic intervention.     

Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis

Membrane-type matrix metalloproteinase 1 (MT1-MMP) plays a critical role in extracellular matrix remodeling under both physiological and pathological conditions. However, the mechanisms controlling its activity on the cell surface remain poorly understood. In this study, we demonstrate that MT1-MMP is regulated by endocytosis. First, we determined that Con A induces proMMP-2 activation in HT1080 cells by shifting endogenous MT1-MMP from intracellular compartments to cell surface. This phenotype was mimicked by the cytoplasmic truncation mutant MT1 Delta C with more robust pro-MMP-2 activation and cell surface expression than wild-type MT1-MMP in transfected cells. MT1 Delta C was subsequently shown to be resistant to Con A treatment whereas MT1-MMP remains competent, suggesting that Con A regulates MT1-MMP activity through cytoplasmic domain-dependent trafficking. Indeed, MT1-MMP was colocalized with clathrin on the plasma membrane and with endosomal antigen 1 in endosomes. Internalization experiments revealed that MT1-MMP is internalized rapidly in clathrin-coated vesicles whereas MT1 Delta C remains on cell surface. Coexpression of a dominant negative mutant of dynamin, K44A, resulted in elevation of MT1-MMP activity by interfering with the endocytic process. Thus, MT1-MMP is regulated by dynamin-dependent endocytosis in clathrin-coated pits through its cytoplasmic domain.     

Human cervical tumor cell (SiHa) surface αvβ3 integrin receptor has associated matrix metalloproteinase (MMP-2) activity

PURPOSE: Integrins are transmembrane heterodimeric molecules that mediate cellular adhesion and are involved in different biological processes, such as tumor development and invasion of tumor cells. Matrixmetalloproteases (MMP) are a family of secreted or membrane proteins capable of digesting extracellular matrix. It has been shown that MMP-2 binds to alphavbeta3 integrin. Recent evidence suggests that a complex of membrane-type MMP (MT1-MMP) and tissue inhibitor of metalloptroteinase-2 (TIMP-2) participate in the activation of alphavbeta3-associated MMP-2. We investigated whether alphavbeta3 and MMP-2 are associated on the membranes of a human cell line, SiHa, and the possible involvement of MT1-MMP and TIMP-2 in the modulation of MMP-2 activity. METHODS: Immunoprecipitation of SiHa membrane extracts with monoclonal antibodies against alphav or MMP-2, and western blots of immunoprecipitates and serum-free conditioned media were performed. TIMP-2 in conditioned medium and MT1-MMP in the membrane fraction was assayed by western blot. Zymography of anti-alphav antibody immunoprecipitates and conditioned media were used to show gelatinolytic activity. RESULTS: The coprecipitation of MMP-2 with alphavbeta3 by anti-alphav antibody is a strong indication that SiHa cell surface alphavbeta3 integrin is a receptor for MMP-2. Immunoblot assays show the expression of MT1-MMP on SiHa cell membranes and secreted TIMP-2 and pro-MMP-2 in the medium. CONCLUSIONS: SiHa cells express all the molecules which are reported to form a complex to activate pro-MMP-2. Active MMP-2 associated with alphavbeta3 may regulate matrix degradation and thereby modulate directed motility of SiHa cells.     

Culture of human cervical cancer cells, SiHa, in the presence of fibronectin activates MMP-2

Purpose: Several studies indicate that integrin receptors are involved in the regulation of matrix metalloproteinase (MMP) expression. Integrin–ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen activated protein kinase pathways. In this present communication, we cultured human cervical cancer cells, SiHa, in the presence of fibronectin to study fibronectin–integrin mediated modulation of MMP activity. Methods: SiHa cells were cultured in serum-free medium (SFCM) in the presence of fibronectin, SFCM was collected and gelatin zymography was performed. Western blot, RT-PCR and immunocytochemistry were performed with SiHa cells cultured in the presence of fibronectin. Results: The culture of SiHa cells in the presence of 50 μg/1.5 ml fibronectin led to expression of pro-MMP-9 and activation of MMP-2 within 2 h. When cells were treated with ERK inhibitor (PD98059) and grown in the presence of fibronectin MMP-2 activation was partially inhibited, but when cells were treated with PI-3K inhibitor (LY294002) and grown in the presence of fibronectin MMP-2 activation was appreciably reduced. Tyrosine phosphorylation of FAK, PI-3K and ERK and nuclear trafficking of ERK were increased in SiHa cells grown in the presence of fibronectin. Increased MT1-MMP mRNA expression and processing of MT1-MMP were also observed in SiHa cells grown in the presence of fibronectin. Conclusions: Our findings indicate that the culture of SiHa cells in SFCM in the presence of fibronectin perhaps generates a signalling cascade which leads to the expression of pro-MMP-9 and the activation of MMP-2 within 2 h. The signalling pathways activated seem to be the FAK/ERK/PI-3K pathway.     

Reactive oxygen species-sensitive p38 MAPK controls thrombin-induced migration of vascular smooth muscle cells

Thrombin has been implicated in the development of atherosclerosis and restenosis, in which migration of vascular smooth muscle cells (VSMC) is a crucial event. Thrombin-stimulated VSMC migration is associated with increased generation of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPKs), and production of growth factors and chemoattractants. In this study, we examined the interrelation of these signals to determine the pathway controlling thrombin-directed migration of human VSMC. Our results show that thrombin stimulated the production of ROS and activation of p38 MAPK. ROS were required for thrombin-induced VSMC migration since both generation of ROS and cell migration were significantly attenuated by inhibitors of NAD(P)H oxidase, diphenyleneiodonium (DPI) and apocynin (Apo.), and by the hydrogen peroxide scavenger, catalase (Cat.). Activation of p38 MAPK by thrombin was inhibited by DPI, Apo. and Cat., indicating ROS are used as messengers for activating this kinase. p38 MAPK is an important step since SB 203580, a selective inhibitor of p38 MAPK, suppressed the cell migration induced by thrombin. Furthermore, thrombin increased the expression of vascular endothelial growth factor (VEGF), a chemoattractant for VSMC, and this expression was inhibited by DPI, Apo., Cat. and SB 203580. Addition of anti-VEGF antibody significantly attenuated thrombin-induced migration. Collectively, the data presented here show that thrombin has stimulated VSMC migration and VEGF expression through an ROS-sensitive p38 MAPK pathway. VEGF synthesized and released by the cell served as a secondary mediator in thrombin-directed migration.     

非类固醇类抗炎药NS398对肺癌H460细胞RECK及MT1-MMP表达的影响

目的 探讨非类固醇类抗炎药NS398对肺癌H460细胞增殖及其Kazal基序逆向诱导半胱氨酸丰富蛋白(RECK)及膜型基质金属蛋白酶1(MT1-MMP)表达的影响.方法 应用MTT方法检测H460细胞生长的抑制率,用免疫荧光法、Western blot法检测RECK及MT1-MMP蛋白的表达.结果 NS398可抑制H460细胞的增殖,促进RECK蛋白的表达,减少MT1-MMP蛋白的含量,并呈剂量依赖关系.结论 NS398可通过促进肺癌细胞RECK的表达并减少MT1-MMP的含量,进而抑制肺癌H460细胞的生长,这可能是肺癌侵袭转移的一个重要机制.     

Glycation cross-links inhibit matrix metalloproteinase-2 activation in vascular smooth muscle cells cultured on collagen lattice

Aims/hypothesis. Extracellular matrix glycation has been proposed to contribute to the arterial stiffness observed in aging and diabetes. We examined whether matrix protein glycation regulates the proleolytic process through the manipulation of matrix metalloproteinases (MMPs) activation, using collagen fibrils model. Methods. Vascular smooth muscle cells were cultured on control or glycated collagen fibrils. Matrix metalloproteinase-2 activation and the production of tissue inhibitors of metalloproteinase (TIMPs) were measured in the conditioned medium by using gelatin zymography and immunoblotting. Membrane type 1 matrix metalloproteinase (MT1-MMP) expression was also measured in cell lysates. Results. When smooth muscle cells were cultured on collagen fibrils, pro-MMP-2 processing to active form was observed in the conditioned medium in coincidence with the increased MT1-MMP expression and the suppressed TIMP-2 production. Culturing smooth muscle cells on glycated collagen fibrils inhibited MMP-2 activation and attenuated MT1-MMP expression without the alteration of TIMP-2 production compared with control fibrils, indicating the possible mechanism of the suppression of MT1-MMP expression for the inhibition of MMP-2 activation on glycated collagen fibrils. Inclusion of aminoguanidine, an inhibitor of cross-linking formation, during collagen glycation restored the MMP-2 activation, suggesting the role of cross-links on the inhibition of MMP-2 activation. Conclusion/interpretation. These observations suggest that glycation-induced cross-linking formation in interstitial collagen contributes to arterial stiffness in aging and diabetes through the manipulation of matrix metalloproteinase activation along with the reduction of the susceptibility to proteolytic enzymes. [Diabetologia (2001) 44: 433–436] Received: 16 October 2000 and in revised form: 18 December 2000     

Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions.

Matrix metalloproteinase-2 (MMP-2) is involved in extracellular matrix remodeling. It is secreted as a proenzyme and activated by membrane type-MMPs (MT-MMP), such as MT1-MMP. In liver fibrosis, MMP-2 is highly expressed in myofibroblasts and may have a profibrogenic role. The mechanisms of its activation in the liver are still unclear. The aim of this work was to show that pro-MMP-2 is efficiently activated in human fibrotic liver and to investigate the role of cell-matrix interactions in this process. Liver specimens obtained from patients with active cirrhosis were compared to normal liver specimens. Human hepatic myofibroblasts were cultured either on plastic, fibronectin, laminin, or on collagen I gels. MMP-2 activity was visualized by gelatin zymography. MMP-2 active form (59 kd) was detected in active cirrhosis but not in normal liver. Myofibroblasts cultured on plastic, fibronectin, or laminin predominantly expressed inactive pro-MMP-2 (66 kd). In contrast, myofibroblasts cultured on collagen I markedly activated the enzyme. Similar results were obtained using membrane fractions from cells previously cultured on collagen or plastic. Activation was inhibited by the tissue inhibitor of metalloproteinases-2 but not by tissue inhibitor of metalloproteinases-1, implicating a MT-MMP-mediated process. Culture on collagen I up-regulated MT1-MMP protein detected by Western blotting, but decreased MT1-MMP mRNA. This study shows that MMP-2 is activated in fibrotic liver. It suggests that interactions between collagen I and myofibroblasts promote this process through a post-translational increase of MT1-MMP expression in these cells.     

反义RNA对胃癌细胞MT1-MMP基因表达和侵袭性的抑制作用

目的:膜研究膜型-1基质金属蛋白酶(MT1- MMP)反义RNA对人胃癌细胞BGC823靶基因表达和侵袭特性的影响方法:利用基因重组技术构建人MT1-MMP反义RNA真核表达载体,转染人胃癌细胞BGC823,应用RT-PCR、MTT、明胶酶谱和体外侵袭实验等方法观察人胃癌细胞BGC823转染前后,MT1-MMP mRNA表达水平、细胞生长、明教酶A活性及细胞体外侵袭能力等指标的变化.结果:成功构建了MT1-MMP反义RNA真核表达载体pasMMP14,将其转染胃癌细胞BGC823后,与阴性对照组相比,实验组MT1- MMP mRNA表达水平降低,抑制率为36%.转染48 h,明教酶A的活化受到了明显抑制.转染72 h,细胞增殖明显受抑(t=2.358,P<0.01 vs空白组:t=2.727 P<0.01 vs阴性组).实验组的穿膜细胞数明显低于空白对照组和阴性对照组(t=5.744,P<0.01;t=5.695,P<0.01).结论:反义RNA对人胃癌细胞MT1-MMP基因表达和侵袭能力具有明显的抑制作用,MT1-MMP基因可作为胃癌抗侵袭治疗的分子靶点.     

Overexpression of MT3-MMP in hepatocellular carcinoma correlates with capsular invasion

BACKGROUND/AIMS: Extracellular matrix-degrading matrix metalloproteinases (MMPs) are invariably up-regulated in epithelial cancers and are key agonists of angiogenesis, invasion and metastasis. Recent studies have shown high levels of various MMPs, including MT1-MMP, MMP-1, MMP-2 and MMP-9, and their involvement in tumor progression in human hepatocellular carcinoma (HCC). However, the expression and role of MT3-MMP in HCC remains unclear. METHODOLOGY: We examined the immunohistochemical expression of MT3-MMP in surgically resected HCCs (n=58), hepatitis C virus (HCV) and hepatitis B virus (HBV)-related chronic hepatitis (n=34) and cirrhosis (n=24). RESULTS: MT3-MMP expression was observed in all non-cancerous liver tissues. In HCCs, 52% (30/58) of patients showed high MT3-MMP expression while the remaining 48% (28/58) of patients showed low expression. A clinicopathological survey demonstrated a significant correlation between high MT3-MMP expression and capsular invasion of carcinoma (p = 0.034) although there was no correlation between high MT3-MMP expression in HCC and overall survival or disease-free survival. CONCLUSIONS: MT3-MMP was expressed not only in chronic hepatitis and liver cirrhosis, but also in HCC, and high MT3-MMP expression correlated significantly with capsular invasion of carcinoma.     

Membrane type 1-matrix metalloproteinase is involved in migration of human monocytes and is regulated through their interaction with fibronectin or endothelium

Membrane type 1-matrix metalloproteinase (MT1-MMP) is involved in endothelial and tumor-cell migration, but its putative role in leukocyte migration has not been characterized yet. Here, we demonstrate that anti-MT1-MMP monoclonal antibody (mAb) impaired monocyte chemotactic protein-1 (MCP-1)-stimulated monocyte migration on fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). In addition, monocyte transmigration through tumor necrosis factor-alpha (TNF-alpha)-activated endothelium is also inhibited by anti-MT1-MMP mAb. Therefore, regulation of MT1-MMP in human peripheral blood monocytes was investigated. First, MT1-MMP clustering was observed at motility-associated membrane protrusions of MCP-1-stimulated monocytes migrating on FN, VCAM-1, or ICAM-1 and at the leading edge, together with profilin, of monocytes transmigrating through activated endothelial cells. In addition, up-regulation of MT1-MMP expression was induced in human monocytes upon attachment to FN in a manner dependent on alpha4beta1 and alpha5beta1 integrins. Binding of monocytes to TNF-alpha-activated human endothelial cells as well as to VCAM-1 or ICAM-1 also resulted in an increase of MT1-MMP expression. These findings correlated with an enhancement of MT1-MMP fibrinolytic activity in monocytes bound to FN, VCAM-1, or ICAM-1. Our data show that MT1-MMP is required during human monocyte migration and endothelial transmigration and that MT1-MMP localization, expression, and activity are regulated in monocytes upon contact with FN or endothelial ligands, pointing to a key role of MT1-MMP in monocyte recruitment during inflammation.     

Membrane type 1-matrix metalloproteinase (MT1-MMP) cooperates with sphingosine 1-phosphate to induce endothelial cell migration and morphogenic differentiation

Membrane type 1-matrix metalloproteinase (MT1-MMP) has been suggested to play an important role in angiogenesis, but the mechanisms involved remain incompletely understood. Using an in vitro model of angiogenesis in which cell migration of bovine aortic endothelial cells (BAECs) and their morphogenic differentiation into capillary-like structures on Matrigel are induced by overexpression of MT1-MMP, we show that the platelet-derived bioactive lipid sphingosine 1-phosphate (S1P) is the predominant serum factor essential for MT1-MMP-dependent migration and morphogenic differentiation activities. In the presence of S1P, MT1-MMP-dependent cell migration and morphogenic differentiation were inhibited by pertussis toxin, suggesting the involvement of Gi-protein-coupled receptor-mediated signaling. Accordingly, cotransfection of BAECs with MT1-MMP and a constitutively active Galphai2 (Q205L) mutant increased cell migration and morphogenic differentiation, whereas treatment of BAECs overexpressing MT1-MMP with antisense oligonucleotides directed against S1P1 and S1P3, the predominant S1P receptors, significantly inhibited both processes. These results demonstrate that MT1-MMP-induced migration and morphogenic differentiation involve the cooperation of the enzyme with platelet-derived bioactive lipids through S1P-mediated activation of Galphai-coupled S1P1 and S1P3 receptors. Given the important contribution of platelets to tumor angiogenesis, the stimulation of endothelial MT1-MMP function by S1P may thus constitute an important molecular event linking hemostasis to angiogenesis.     

Role of the JNK pathway in thrombin-induced ICAM-1 expression in endothelial cells

OBJECTIVE: Thrombin induces leukocyte adherence to endothelial cells via increased expression of intercellular adhesion molecule-1 (ICAM-1). Although ICAM-1 expression is regulated by NF-kappaB, recent studies have suggested that additional signaling mechanisms may also be involved. The goal of this study was to determine whether mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38), mediate thrombin-induced ICAM-1 expression in endothelial cells. METHODS: Western blot analysis using anti-ICAM-1 antibody and luciferase assays were performed in cultured endothelial cells after addition of signal transduction inhibitors or transfection of various gene constructs. JNK kinase activity was determined by a kinase assay using c-Jun as a substrate or by Western blot analysis with anti-phospho-JNK antibody. RESULTS: Treatment of endothelial cells with the JNK-specific inhibitors, SP600125 or JNK inhibitory peptide 1 (JNKI1), resulted in a significant decrease in thrombin-induced ICAM-1 expression as demonstrated by Western blot analysis (67 +/- 3% and 72 +/- 7%, respectively). In contrast, inhibitors of MEK and p38 had only minimal effect. The combination of SP600125 and the NF-kappaB inhibitor, BAY11-7082, resulted in complete inhibition of thrombin-induced ICAM-1 expression. The Galpha(q) inhibitor, YM-254890, inhibited thrombin-induced JNK activation and ICAM-1 expression. Dominant-negative Ras and Rac1, but not Rho, inhibited thrombin-induced JNK activation and ICAM-1 promoter activity. Finally, thrombin-induced JNK activation and ICAM-1 promoter activity were inhibited by betaARK1ct (a Gbetagamma subunit scavenger) and Csk. CONCLUSIONS: These data suggest that, in concert with NF-kappaB, JNK regulates thrombin-induced ICAM-1 expression by a mechanism that is dependent on Galpha(q), Gbetagamma, Ras, Rac1 and the Src kinase family.