Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic ␤ 2 receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade.The potent vasoactive peptide angiotensin II (Ang II) 1 is also an important regulator of cellular proliferation and hypertrophy. This peptide binds to two main subtypes of receptors (AT1 and AT2) that both belong to the superfamily of G proteincoupled receptors (GPCR) but display opposite biological and physiological effects. The AT1 receptor mediates most of the known cardiovascular and central actions of Ang II. This subtype has mitogenic and trophic effects in many tissues and cell types and transduces multiple intracellular signaling cascades typically associated with GPCR activation. In contrast, AT2 behaves like a "natural antagonist" of the AT1 subtype on most physiological functions, and induces anti-proliferative and proapoptotic effects in vitro and in vivo (for reviews, see Refs. 1-5).The AT2 receptor activates unconventional signaling pathways that in most cases do not involve coupling to classical regulatory G proteins. A growing body of evidence indicates that anti-growth effects of the AT2 receptor are associated with activation of tyrosine phosphatases and inhibition of protein kinases, which ultimately lead to inhibition of extracellular regulated kinase (ERK2). In many cell types, AT2 is functionally coupled to the Src homology 2 domain-containing tyrosine phosphatase SHP-1 (6 -8). This phosphatase has been shown to play a central role in the AT2 signaling cascades leading to inhibition of AT1-induced PYK2 and Jun kinase (9), AT1-transactivated EGF receptor tyrosine kinase (10), and insulin-induced phosphatidylinositol 3-kinase and Akt activation (11).AT2 negatively cross-talks with receptor tyrosine kinases (RTK) such as bFGF, EGF, and insulin receptors (10, 12, 13) by targeti...

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Background-Tissue factor (TF), the surface receptor for the serine protease factor VIIa (FVIIa) and the initiator of the extrinsic coagulation cascade, supports vessel development and tumor metastasis by activation of extracellular, protease-dependent signaling pathways. The molecular mechanisms that do not require proteolytic activity of FVIIa are not yet known. The aim of the study, therefore, was to investigate the effects of active-site-inhibited FVIIa (FFR-FVIIa) on TF-mediated signaling. Methods and Results-After stimulation with FVIIa and FFR-FVIIa, migration and activation of the GTPase Rac (Rac1) or the mitogen-activated protein kinase p38 (p38) were analyzed in J82 cells. FVIIa and FFR-FVIIa stimulated migration and activation of Rac1 and p38 in a TF-specific, dose-and time-dependent manner. Enhancement of migration required activation of Rac1 and p38, because it was abolished after inhibition with SB203580 or overexpression of dominant negative p38 and Rac1. The cytoplasmic domain of TF was necessary because no effects of FFR-FVIIa could be detected after transfection of a TF deletion mutant lacking the cytoplasmic domain. Conclusions-We

Objective-The serine protease MT-SP1/matriptase plays an important role in cell migration and matrix degradation.Hepatocyte growth factor (HGF), urokinase-type plasminogen activator (uPA), and protease-activated receptor 2 (PAR-2) have been identified as in vitro substrates of MT-SP1/matriptase. Because PAR-2 is expressed in endothelial cells and contributes to inflammatory processes, we sought to investigate the effects of MT-SP1/matriptase on endothelial cytokine expression and analyzed MT-SP1/matriptase expression in vascular cells and atherosclerotic lesions. Methods and Results-In endothelial cells, recombinant MT-SP1/matriptase dose-dependently induced interleukin (IL)-8and IL-6 mRNA and protein expression dependent on its proteolytic activity. MT-SP1/matriptase time-dependently induced phosphorylation of p38 MAPK and p42/44 MAPK. Inhibitor experiments revealed that p38 MAPK and PKC␣ were necessary for IL-8 induction. PAR-2 downregulation abolished and PAR-2 overexpression augmented MT-SP1/ matriptase-induced IL-8 expression as evidence for PAR-2 signaling. In human atherectomies, MT-SP1/matriptase was expressed in blood cells adherent to the endothelium. Concordantly, basal MT-SP1/matriptase expression was detected in isolated monocytes. Coincubation of monocytes and endothelial cells resulted in an increased IL-8 release, which was reduced after downregulation of endothelial PAR-2 and monocytic MT-SP1/matriptase. Key Words: pathophysiology Ⅲ growth factors Ⅲ endothelium M T-SP1/matriptase is a trypsin-like, multi-domain serine protease expressed primarily in epithelial cells. [1][2][3][4] Its importance in the biology of surface-lining epithelial cells became apparent in MT-SP1/matriptase knockout mice presenting with a severe deficient epidermal barrier function as well as abnormal hair follicle development and disturbed thymic homeostasis. 5 Moreover, MT-SP1/matriptase is upregulated in different malignant tissues 6 -8 and may be expressed in microvascular endothelial cells. 9 Besides its N-terminal transmembrane signal anchor MT-SP1/matriptase contains two putative regulatory modules: 2 tandem repeats of a CUB domain (C1r/s, Uegf, Bone morphogenetic protein-1) and 4 tandem repeats of a low density lipoprotein (LDL) receptor domain. 1,4 The C-terminal serine protease domain consists of a catalytic triad comprising His-57, In addition to the membrane-anchored form of MT-SP1/matriptase, a soluble form of the protease has been identified lacking the N-terminal 172 amino acids. 1 Shedding from the extracellular surface 11,12 or alternative splicing 3,10 may be the mechanisms leading to the truncated form of MT-SP1/matriptase isolated from human milk. 13 Cleavage within its activation motif generates the 2-chain active protease from a single-chain zymogen. The activation of MT-SP1/matriptase requires its cognate Kunitz-type inhibitor hepatocyte growth factor activator inhibitor (HAI)-1, its noncatalytic domains as well as its serine protease domain. 14 Three macromolecular substrates of MT-SP1/matriptase h...

Objective-In acute myocardial infarction (AMI), proinflammatory plasma C-reactive protein values are strongly associated with postinfarction morbidity and mortality. So far, the cause of these inflammatory changes is not well understood. Therefore, we sought to investigate the relationship between the activation of coagulation and subsequent systemic inflammatory changes in AMI. Methods and Results-Factor Xa (FXa) bound to tissue factor pathway inhibitor and prothrombin fragments F1ϩ2 (F1ϩ2) were used as a measure for activated coagulation. To assess systemic inflammatory changes, plasma interleukin (IL)-6 and IL-8 concentrations were analyzed by immunoassay. Blood samples were taken from 21 patients with AMI and 20 patients with stable angina pectoris. In AMI, tissue factor pathway inhibitor FXa but not F1ϩ2 plasma levels were associated with circulating IL-8 (Pϭ0.01). In vitro experiments revealed that FXa stimulated IL-8 and monocyte chemoattractant protein-1 release and RNA expression in endothelial cells and mononuclear leukocytes by activation of protease-activated receptor-1. Key Words: coagulation Ⅲ myocardial infarction Ⅲ inflammation Ⅲ cytokines T he main initiator of the extrinsic coagulation cascade is tissue factor (TF), the receptor and cofactor for plasma coagulation factor VII/VIIa. Under physiological conditions TF is mainly expressed at extravascular sites. However, TF is induced by several inflammatory mediators such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1. 1,2 In acute myocardial infarction (AMI), disruption of atherosclerotic plaques exposes TF-positive cells within the plaque-to-plasma clotting factors and initiates local thrombosis with subsequent occlusion of the coronary vessel. 3 In addition, increased TF expression occurs on circulating monocytes and microparticles in acute coronary syndromes and may thereby contribute to activation of coagulation. 4 -6 A soluble form of TF within the circulating blood may also support coronary thrombosis. 7 Stimulation of the TF-thrombin pathway does not only occur at the site of the plaque but also within the ischemic myocardium where activated coagulation factors may enhance inflammatory responses and deteriorate infarct size. 8 The endogenous Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI)-1 inhibits initiation of TF-induced blood coagulation. TFPI binds and inactivates factor Xa (FXa). The TFPI-FXa complex then binds and inactivates FVIIa. Increased levels of the TFPI-FXa complex may reflect both increased FXa generation and increased TFPI concentrations. 9 In addition to the full length TFPI, most of the plasma TFPI circulates in truncated forms that are bound to plasma lipoproteins. These truncated forms lack their C-terminal domains and exhibit reduced affinity for vascular wall proteolysis. Conclusion-OurBinding of the serine protease FVII to TF results in generation of the coagulation protease FXa and subsequently thrombin, both known to induce cell signaling. FXa shows dose-dependent inductio...

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Abstract-Tissue factor (TF), the cell surface receptor for the serine protease FVIIa supports cell migration by interaction with the cytoskeleton. Intracellular signaling pathways dependent on the cytoplasmic domain of TF modify cell migration and may alter vascular remodeling. Key Words: arterial injury Ⅲ tissue factor Ⅲ smooth muscle cells Ⅲ restenosis T issue factor (TF) is a transmembrane glycoprotein that initiates the clotting cascade and is considered to be a major regulator of coagulation and hemostasis. Previous studies have shown that TF is important in mediating acute arterial thrombosis and intimal hyperplasia in several animal models. [1][2][3][4] Previous studies suggest an additional role for TF in tumor metastasis, angiogenesis, and cell migration. [5][6][7][8][9] After binding TF FVII is activated to FVIIa, a serine protease. The complex TF-FVIIa induces gene transcription and activates signaling cascades. 10 -14 Independent of the proteolytic activity TF ligand binding supports cell spreading and migration. 5,15 Furthermore, phosphorylation of the cytoplasmic domain serves as a regulator for angiogenic responses. [7][8][9] Because mice lacking the cytoplasmic domain of TF exhibit normal development we sought to investigate if deletion of the cytoplasmic domain of TF in mice alters vascular responses to arterial injury. Materials and Methods Mouse Injury ModelMice with a deletion of 18 amino acids of the cytoplasmic domain of TF (TF ⌬ct/⌬ct ) were generated by Cre-lox recombination technique on a MF1/129S/v/Swiss strain background. 16 These animals displayed normal embryonic and postnatal development, fertility, and coagulation parameters. Mice were bred and housed under specific pathogen-free conditions. All studies were approved by the institutional Animal Care and Use Committee (Bayerisches Wissenschaftsministerium, München, Germany). Mice underwent carotid ligation and femoral-artery wire injury model as described. 17,18 Before surgery, all mice were anesthetized by intraperitoneal injection with a solution composed of ketamine (80 mg/kg body weight) and xylazine (5 mg/kg body weight) for carotid ligation we used a midline neck incision, the right external carotid artery was looped proximally and tied off distally. Additional silk ties were looped round the common and internal carotid arteries for temporary vascular control during the procedure. For the femoral artery injury model a 0.014-in flexible angioplasty guide wire was introduced into the right femoral artery and endothelial denudation injury was performed using wire withdrawal injury and 3 passes along the artery. After removal of the wire the arteriotomy was closed and normal blood flow restored. At the time of euthanasia (14 days for carotid ligation and 28 days for femoral wire injury), the animals were reanesthetized, and after an overdose of pentobarbital (210 mg/kg IP) in situ perfusion fixation was achieved with phosphatebuffered paraformaldehyde (4%, 0.1 mol/L sodium phosphate buffer, pH 7.3). Both injured right and uninjure...

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