Takehiko Tarui

Plasmin-induced migration requires signaling through protease-activated receptor 1 and integrin α9β1

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 α9β1. Here we report that plasmin specifically interacts with α9β1 and that plasmin induces of cells expressing migration recombinant α9β1 (α9-Chinese hamster ovary (CHO) cells). Migration was dependent on an interaction of the kringle domains of plasmin with α9β1 as well as the catalytic activity of plasmin. Angiostatin, representing the kringle domains of plasmin, alone did not induce the migration of α9-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 α9-CHO cells. These results suggest that plasmin induces migration by kringle-mediated binding to α9β1 and simultaneous proteolytic activation of PAR-1.

Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin αvβ3 induces signal transduction and enhances plasminogen activation

It has been questioned whether there are receptors for urokinase- type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation.These uPA-induced signaling events are not mediated by uPAR,but mediated by unidentified,lower-affinity receptors for the uPA kringle.We found that uPA binds specifically to integrin αvβ3 on CHO cells depleted of uPAR.The binding of uPA to αvβ3 required the uPA kringle domain. The isolated uPA kringle domain binds specifically to purified,recombinant soluble, and cell surface αvβ3, and other integrins ( α4β1 and α9β 1), and induced migration of CHO cells in an αvβ3-dependent manner.The binding of the uPA kringle to αvβ3 and uPA kringle-induced αvβ-dependent cell migration were blocked by homologous plasminogen kringles 1–3 or 1–4 (angiostatin), a known integrin antagonist. We studied whether the binding of uPA to integrin αvβ3 through the kringle domain plays a role in plasminogen activation. On CHO cell depleted of uPAR, uPA enhanced plasminogen activation in a kringle and αvβ3-dependent manner.Endothelial cells bound to and migrated on uPA and uPA kringle in an αvβ3-dependent manner.These results suggest that uPA binding to integrins through the kringle domain plays an important role in both plasminogen activation and uPA-induced intracellular signaling. The uPA kringle-integrin interaction may represent a novel therapeutic target for cancer, inflammation, and vascular remodeling.

The Role of the CPNKEKEC Sequence in the β2 Subunit I Domain in Regulation of Integrin αLβ2 (LFA-1)

The αL I (inserted or interactive) domain of integrin αLβ2 undergoes conformational changes upon activation. Recent studies show that the isolated, activated αL I domain is sufficient for strong ligand binding, suggesting the β2 subunit to be only indirectly involved. It has been unclear whether the activity of the αL I domain is regulated by the β2 subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169–176) in the β2 I domain with a corresponding β3 sequence, or mutating Lys174 to Thr, constitutively activates αLβ2 binding to ICAM-1. These mutants do not require Mn2+ for ICAM-1 binding and are insensitive to the inhibitory effect of Ca2+. We have also localized a component of the mAb 24 epitope (a reporter of β2 integrin activation) in the CPNKEKEC sequence. Glu173 and Glu175 of the β2 I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon β2 integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the αL I domain did not eliminate the mAb 24 epitope. This confirms that the αL I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the β2 subunit I domain. These results suggest that the CPNKEKEC sequence of the β2 I domain is involved in regulating the αL I domain.

Attachment of Human Colon Cancer Cells to Vascular Endothelium Is Enhanced by N-Acetylglucosaminyltransferase V

Expression of N-acetylglucosaminyltransferase V (GnT-V) in colon cancer has been shown to be related to hematogenous metastasis and poor prognosis. To investigate the mechanism by which cancer cells expressing GnT-V metastasize to distant organs, we established GnT-V-overexpressing DLD-1 and WiDr cells (human colon cancer cell lines) by transfecting them with a GnT-V expression vector. Attachment to endothelial cells expressing E-selectin was studied, and expression of the E-selectin ligand, sialyl Lewis x, in colon cancer cells was investigated. Both of the cell lines showed reduced adhesion to fibronectin as compared with mock transfectants. In contrast, attachment to human umbilical vein endothelial cells expressing E-selectin was significantly enhanced by GnT-V expression (p < 0.01). Sialyl Lewis x is a ligand for E-selectin and a marker for poor prognosis of colon cancer. Its synthesis in cells has been shown to involve GnT-V. We demonstrated that expression of sialyl Lewis x in colon cancer cells was induced by GnT-V expression. These results suggest that GnT-V induces sialyl Lewis x expression and leads colon cancer cells to metastasize by enhancing their ability to attach to vascular endothelium in distant organs, such as liver or lung. Inhibition of GnT-V activity may prevent metastasis in colon cancer patients with high sialyl Lewis x expression.