In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is ␤1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing ␣v␤3 or certain ␤1 integrin heterodimers (␣3␤1, ␣4␤1, ␣5␤1, ␣6␤1, ␣9␤1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates ␣3␤1-dependent migration on laminin 5. Expression of TF suppresses ␣3␤1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of ␣3␤1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra-and extracellular interactions and phosphorylation regulates TF's function in cell motility. INTRODUCTIONTissue factor (TF) is the cell surface receptor for the serine protease coagulation factor VIIa (VIIa; Ruf and Edgington, 1994). The complex of TF-VIIa activates the coagulation cascade, leading to thrombin generation, fibrin formation, and platelet activation. Local fibrin deposition is frequently observed in malignancy and TF plays an important role in cancer invasion and metastasis (Dvorak et al., 1992;Shoji et al., 1998). TF supports metastatic tumor dissemination (Mueller et al., 1992) by fibrin-dependent pathways (Palumbo et al., 2000), by aiding thrombin-dependent tumor cell survival (Fischer et al., 1995;Ruf and Mueller, 1996;Zain et al., 2000), and through signaling that involves the short TF cytoplasmic domain (Bromberg et al., 1995;Mueller and Ruf, 1998). TF is also found at the leading edge of invasive tumors (Fischer et al., 1999) and in angiogenic endothelial cells (Contrino et al., 1996). In vitro studies documented a close association of TF with cytoskeletal structures (Carson et al., 1994;Ott et al., 1998;Mü ller et al., 1999) and indicated potential roles in regulating cell motility, such as reverse endothelial cell migration of monocytes (Randolph et al., 1998), enhanced chemotactic fibroblast migration (Siegbahn et al., 2000), and tumor cell adhesion and migration on extracellular ligands for TF (Ott et al., 1998;Fischer et al., 1999). In TF cytoplasmic domain-deleted mice, we have recently provided evidence that the TF cytoplasmic domain can negatively regulate angiogenesis and endothelial sprouting (Belting et al., 2004). However, the molecular intera...

Angiostatin, the N-terminal four kringles (K1-4) of plasminogen, blocks tumor-mediated angiogenesis and has great therapeutic potential. However, angiostatin's mechanism of anti-angiogenic action is unclear. We found that bovine arterial endothelial (BAE) cells adhere to angiostatin in an integrin-dependent manner and that integrins ␣ v ␤ 3 , ␣ 9 ␤ 1 , and to a lesser extent ␣ 4 ␤ 1 , specifically bind to angiostatin. ␣ v ␤ 3 is a predominant receptor for angiostatin on BAE cells, since a functionblocking antibody to ␣ v ␤ 3 effectively blocks adhesion of BAE cells to angiostatin, but an antibody to ␣ 9 ␤ 1 does not. ⑀-Aminocaproic acid, a Lys analogue, effectively blocks angiostatin binding to BAE cells, indicating that an unoccupied Lys-binding site of the kringles may be required for integrin binding. It is known that other plasminogen fragments containing three or five kringles (K1-3 or K1-5) have an anti-angiogenic effect, but plasminogen itself does not. We found that K1-3 and K1-5 bind to ␣ v ␤ 3 , but plasminogen does not. These results suggest that the anti-angiogenic action of angiostatin may be mediated via interaction with ␣ v ␤ 3 . Angiostatin binding to ␣ v ␤ 3 does not strongly induce stress-fiber formation, suggesting that angiostatin may prevent angiogenesis by perturbing the ␣ v ␤ 3 -mediated signal transduction that may be necessary for angiogenesis.Angiogenesis is a complex multistep process that includes endothelial cell proliferation, migration, and differentiation, degradation of extracellular matrices, tube formation, and sprouting of new capillary branches. Several recent studies show that tumor growth and metastasis are angiogenesis-dependent (1-6). Tumors often overexpress several pro-angiogenic molecules, including members of the fibroblast growth factor (7) and the vascular endothelial growth factor/vascular permeability factor families (8, 9). Excessive angiogenesis is a part of the pathology of cancer, and preventing this process is a promising therapeutic strategy.A number of fragments or cryptic domains of large protein molecules have been identified as angiogenesis inhibitors. Angiostatin, a proteolytic fragment of plasminogen (1), inhibits proliferation (10), blocks migration (11), and increases apoptosis (12) of endothelial cells. It has been reported that angiostatin binds to ATP synthase on the cell surface (13) and that the binding of angiostatin to ATP synthase is related to angiostatin's anti-angiogenic effect (e.g. the down-regulation of endothelial cell proliferation) (13). Likewise, endostatin (a proteolytic fragment of collagen XVIII) (14), the 16-kDa N-terminal fragment of prolactin (15), an N-terminally truncated platelet factor 4 (16), and a C-terminal fragment of matrix metalloprotease 2 named PEX (17) are potent angiogenesis inhibitors. Recently, several non-collagenous domains from collagen type IV (canstatin, arrestin, and tumstatin) have been shown to have anti-angiogenic activities (18 -21). These endogenous angiogenesis inhibitors have a lot of therapeutic p...

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins ␣4␤1, ␣6␤1, ␣9␤1, and ␣v␤3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to ␣4␤1 and ␣v␤3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein.In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.

BackgroundSepsis is a leading cause of death and long-term disability in developed countries. A comprehensive report on the incidence, clinical characteristics, and evolving management of sepsis is important. Thus, this study aimed to evaluate the characteristics, management, and outcomes of patients with severe sepsis in Japan.MethodsThis is a cohort study of the Focused Outcomes Research in Emergency Care in Acute Respiratory Distress Syndrome, Sepsis, and Trauma (FORECAST) study, which was a multicenter, prospective cohort study conducted at 59 intensive care units (ICUs) from January 2016 to March 2017. We included adult patients with severe sepsis based on the sepsis-2 criteria.ResultsIn total, 1184 patients (median age 73 years, interquartile range (IQR) 64–81) with severe sepsis were admitted to the ICU during the study period. The most common comorbidity was diabetes mellitus (23%). Moreover, approximately 63% of patients had septic shock. The median Sepsis-related Organ Failure Assessment (SOFA) score was 9 (IQR 6–11). The most common site of infection was the lung (31%). Approximately 54% of the participants had positive blood cultures. The compliance rates for the entire 3-h bundle, measurement of central venous pressure, and assessment of central venous oxygen saturation were 64%, 26%, and 7%, respectively. A multilevel logistic regression model showed that closed ICUs and non-university hospitals were more compliant with the entire 3-h bundle. The in-hospital mortality rate of patients with severe sepsis was 23% (21–26%). Older age, multiple comorbidities, suspected site of infection, and increasing SOFA scores correlated with in-hospital mortality, based on the generalized estimating equation model. The length of hospital stay was 24 (12–46) days. Approximately 37% of the patients were discharged home after recovery.ConclusionOur prospective study showed that sepsis management in Japan was characterized by a high compliance rate for the 3-h bundle and low compliance rate for central venous catheter measurements. The in-hospital mortality rate in Japan was comparable to that of other developed countries. Only one third of the patients were discharged home, considering the aging population with multiple comorbidities in the ICUs in Japan.Trial registrationUMIN-CTR, UMIN000019742. Registered on 16 November 2015.Electronic supplementary materialThe online version of this article (10.1186/s13054-018-2186-7) contains supplementary material, which is available to authorized users.

Neocortical projection neurons arise from a pseudostratified ventricular epithelium (PVE) from embryonic day 11 (E11) to E17 in mice. The sequence of neuron origin is systematically related to mechanisms that specify neuronal class properties including laminar fate destination. Thus, the neurons to be assembled into the deeper layers are the earliest generated, while those to be assembled into superficial layers are the later generated neurons. The sequence of neuron origin also correlates with the probability of cell cycle exit (Q) and the duration of G1-phase of the cell cycle (T(G1)) in the PVE. Both Q and T(G1) increase as neuronogenesis proceeds. We test the hypothesis that mechanisms regulating specification of neuronal laminar destination, Q and T(G1) are coordinately regulated. We find that overexpression of p27(Kip1) in the PVE from E12 to E14 increases Q but not T(G1) and that the increased Q is associated with a commensurate increase in the proportion of exiting cells that is directed to superficial layers. We conclude that mechanisms that govern specification of neocortical neuronal laminar destination are coordinately regulated with mechanisms that regulate Q and are independent of mechanisms regulatory to cell cycle duration. Moreover, they operate prior to postproliferative mechanisms necessary to neocortical laminar assembly.

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are ␣ 9 ␤ 1 and ␣ v ␤ 3 , and they bind in an RGDindependent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin ␣ 9 ␤ 1 and ␣ v ␤ 3 recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg 481 and the Asp-Leu-Pro-Glu-Phe residues (residues 488 -492) were critical for ␣ 9 ␤ 1 binding, but the RGD motif (residues 484 -486) was not. In contrast, the RGD motif was critical for ␣ v ␤ 3 binding, but the other residues flanking the RGD motif were not. As the RX 6 DLPEF ␣ 9 ␤ 1 recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that ␣ 9 ␤ 1 may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that ␣ 9 ␤ 1 bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin ␣ 9 ␤ 1 , applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and ␣ 9 ␤ 1 have similar binding specificities for ADAMs and that ␣ 9 ␤ 1 , or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As ␣ 9 ␤ 1 is a receptor for many ADAM disintegrins and ␣ 9 ␤ 1 and ADAMs are widely expressed, ␣ 9 ␤ 1 -ADAM interaction may be of a broad biological importance. ADAMs1 (a disintegrin and metalloproteases) or MDC (metalloprotease/disintegrin/cysteine-rich) proteins are a family of transmembrane glycoproteins of more than 30 members (see www.people.virginia.edu/ϳjag6n/Table_of_the_ADAMs.html and www.gene.ucl.ac.uk/nomenclature/genefamily/metallo. html). ADAMs have a prodomain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, an EGF-like domain, a transmembrane domain, and a cytoplasmic tail (1-3). Several ADAMs are involved in crucial biological processes such as fertilization (ADAM1, 2, and 3) (4 -6) and muscle cell differentiation (meltrin-␣, ADAM12) (7, 8). Most, but not all, ADAMs have a catalytically active metalloprotease domain, which processes several biologically important cell surface proteins including tumor necrosis factor-␣ (tumor necrosis factor converting enzyme (TACE), ADAM17) (for reviews, see Refs. 9 and 10), Alzheimer protein precursor (ADAM10 and 17) (11, 12), Delta (ADAM10) (9, 13, 14), and heparin-binding EGF (ADAM9) (15).The ADAM disintegrin domains are homologous to snake ven...

Urokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement. uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear. uPAR is a ligand for several integrins (e.g. ␣ 5 ␤ 1 ) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to ␣ 5 ␤ 1 in cis is involved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-␣ 5 antibody, RGD peptide, and function-blocking mutations in ␣ 5 ␤ 1 blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and ␣ 5 ␤ 1 , but ␣ 5 ␤ 1 alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for ␣ 5 ␤ 1 to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to ␣ 5 ␤ 1 in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA⅐uPAR complex to ␣ 5 ␤ 1 via uPAR, and (c) signal transduction through ␣ 5 ␤ 1 .

Plasmin is a major extracellular protease that elicits intracellular signals to mediate platelet aggregation, chemotaxis of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types in a G protein-dependent manner. Angiostatin, a fragment of plasmin(ogen), is a ligand and an antagonist for integrin ␣ 9 ␤ 1 . Here we report that plasmin specifically interacts with ␣ 9 ␤ 1 and that plasmin induces migration of cells expressing recombinant ␣ 9 ␤ 1 (␣ 9 -Chinese hamster ovary (CHO) cells). Migration was dependent on an interaction of the kringle domains of plasmin with ␣ 9 ␤ 1 as well as the catalytic activity of plasmin. Angiostatin, representing the kringle domains of plasmin, alone did not induce the migration of ␣ 9 -CHO cells, but simultaneous activation of the G protein-coupled protease-activated receptor (PAR)-1 with an agonist peptide induced the migration on angiostatin, whereas PAR-2 or PAR-4 agonist peptides were without effect. Furthermore, a small chemical inhibitor of PAR-1 (RWJ 58259) and a palmitoylated PAR-1-blocking peptide inhibited plasmin-induced migration of ␣ 9 -CHO cells. These results suggest that plasmin induces migration by kringle-mediated binding to ␣ 9 ␤ 1 and simultaneous proteolytic activation of PAR-1.