In some tests, migration of uPAR-overexpressing MCF-7 cells was quantitated by repairing the membranes in methanol and staining the migratory cells with 0

In some tests, migration of uPAR-overexpressing MCF-7 cells was quantitated by repairing the membranes in methanol and staining the migratory cells with 0.1% crystal violet. least 6 h. The uPA-induced upsurge in MCF-7 cell migration was noticed selectively on vitronectin-coated areas and was mediated by way of a 1-integrin (most likely V1) and V5. When MCF-7 cells had been transfected expressing V3and treated with uPA, ERK was phosphorylated; nevertheless, the cells didn’t demonstrate elevated migration. Neutralizing the function of V3, with preventing Aminoguanidine hydrochloride Aminoguanidine hydrochloride antibody, restored the power of to market cellular migration uPA. Thus, we’ve showed that uPA promotes mobile migration, within an integrin-selective way, by initiating a uPAR-dependent signaling cascade where Ras, MEK, ERK, and MLCK serve as important downstream effectors. Keywords:urokinase-type plasminogen activator, myosin light string kinase, integrins, vitronectin, mobile migration Urokinase-type plasminogen activator (uPA)1is a serine proteinase that is synthesized as a 54-kD single chain (sc) zymogen and converted into the active two chain form (tcuPA) by numerous proteinases, Rabbit Polyclonal to STEA3 including plasmin (de Munk and Rijken 1990). Both scuPA and tcuPA bind with high affinity (Kdof 0.11.0 nM) to the cell-surface receptor, uPAR (Ellis and Dano 1992). Binding to uPAR may cause scuPA to express intrinsic proteinase activity (Manchanda and Schwartz 1991;Wang et al. 1997), promote the conversion of scuPA to tcuPA (Ellis et al. 1989), and accelerate the activation of cell-associated plasminogen (Cohen et al. 1991;Ellis and Dano 1992). These reactions increase cell-surface proteinase activity, which may be important for malignancy cell invasion and metastasis in vivo (for evaluate seeAndreasen et al. 1997). uPAR is usually linked to the cell surface by a glycosyl-phosphatidylinositol (GPI) anchor (Ploug et al. 1991) and thus lacks transmembrane and intracytoplasmic domains. Nevertheless, uPAR expresses multiple activities, unrelated to proteinase localization, which regulate the conversation of cells with the microenvironment. First, uPAR binds directly to vitronectin and in some cells may augment or substitute for the function of V-containing integrins (Wei et al. 1994). uPAR also associates with integrins (Bohuslav et al. 1995;Simon et al. 1996;Sitrin et al. 1996;Wei et al. 1996;Xue et al. 1997) and may, by direct conversation, modulate the function of specific 1and 2subunitcontaining integrins (Wei et al. 1994;Sitrin et al. 1996;May et al. 1998). Finally, uPAR functions as a signaling receptor, probably with the aid of a transmembrane adaptor protein or multiprotein-complex which includes integrins (Chapman 1997). The signaling cascades that are activated when uPA binds to uPAR are only partially characterized. Known protein tyrosine kinases in theSrc-family, including p60fyn, p53/p56lyn, p56/p59hck, and p59fgr, have been coimmunoprecipitated with uPAR (Dumler et al. 1993,Dumler et al. 1998;Bohuslav et al. 1995;Resnati et al. 1996;Konakova et al. 1998) and at least p56/p59hckis activated by uPA (Resnati et al. 1996;Konakova et al. 1998). uPA also activates protein kinase C (Busso et al. 1994), the JAK1/STAT1 signaling pathway (Koshelnick et al. 1997;Dumler et al. 1998), focal adhesion kinase (Tang et al. 1998), and the mitogen activated protein (MAP) kinases, extracellular-signal regulated kinase (ERK) 1 and 2 (Kanse et al. 1997;Konakova et al. 1998;Nguyen et al. 1998;Tang et al. 1998). The proteolytic activity Aminoguanidine hydrochloride of uPA is not required for these signal transduction responses. uPA promotes the migration of diverse cell types (Odekon et al. 1992;Busso et al. 1994;Resnati et al. 1996;Stefansson and Lawrence 1996;Nguyen et al. 1998); however, it is not clear how the numerous activities of the uPA/uPAR system are integrated to generate this response. In MCF-7 breast malignancy cells, activation of ERK is usually apparently essential for uPA-promoted cellular migration since the MAP kinase kinase (MEK) inhibitor, PD098059, completely blocks the uPA response without affecting Aminoguanidine hydrochloride the basal level of cellular migration (Nguyen et al. 1998). Once activated, ERK may translocate to the nucleus and regulate gene expression by modifying transcription factors (Gille et al. 1992;Marais et al. 1993;Hipskind et al. 1994;Hill and Treisman 1995); however, ERK also modifies multiple cytoplasmic substrates (Kanemitsu and Lau 1993;Lin et al. 1993;Klemke et al. 1997).Klemke et al. 1997demonstrated that ERK phosphorylates and thereby activates myosin light chain kinase (MLCK), promoting cellular migration. Activated ERK also suppresses the function of specific integrins (Hughes et al. 1997) and may promote quick disassembly of focal contacts (Xie et al. 1998), allowing more effective cytoskeletal contraction at the rear of the migrating cell (for review seeLauffenburger and Horwitz 1996). The goal of this.