Cloning, nucleotide sequencing and expression of cDNAs encoding mouse urokinase‐type plasminogen activator

Abstract

Controlled extracellular proteolysis is catalyzed in part by the secretion of plasminogen activators. As a step in the study of the expression of these enzymes in mouse tissues, we have isolated five cDNAs encoding the mouse urokinase-type plasminogen activator from a cDNA library prepared with size-selected mRNA from MSVtransformed 3T3 cells. The longest cDNA insert contains the entire coding region of mouse urokinase, 58 base pairs of the 5' non-coding region, and the entire 3' non-coding region, which is 942 base pairs long. The deduced protein sequence, which starts with a signal peptide of 20 amino acids, shows extensive homology to that of human and porcine urokinase. However, in contrast to these enzymes, mouse urokinase contains no N-glycosylation site. Bacteria harbouring one of the recombinant plasmids synthesize and secrete into their periplasm a protease indistinguishable from mouse urokinase.Plasminogen activators (PAS) are serine proteases of trypsin-like specificity, which convert plasminogen, an inactive zymogen present in plasma and most other extracellular fluids, into plasmin, a neutral protease of broad specificity PAS are synthesized by a number of cell types, usually under a strict and cell-specific hormonal control. Enzyme production has been correlated with several processes involving cell migration and tissue remodeling [9]. PA induction can result from a selective increase in either urokinase [lo, 111 or TA [12] mRNA content. In addition, PA activity can be modulated at post-translational levels, which include the secretion of the enzyme [l 11 and the concomitant production of PA-specific protease inhibitors [13, 141. In the mouse, PA production has been studied in a variety of physiological and pathological situations, including embryonic development [4, 151 and inflammation [ 161. However, no information has been available as to the primary structure of the mouse enzymes, and the probes required to study the expression of the mouse PA genes have not yet been described. We report here the isolation and characterization of cDNA clones containing the entire coding sequence of mouse urokinase mRNA.

Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice

“…The murine uPA gene (including its signal peptide) was isolated from plasmid pDB1519, 34 restricted by XbaI and NsiI, and residues 128 to 135 followed by a stop codon were reintroduced as a synthetic fragment. The ATF gene was then inserted between the CMV immediate-early promoter (extending from position −603 to −15) and the SV40 late polyadenylation signal sequence.…”

“…The membranes were prehybridized with denatured sonicated salmon sperm DNA (100 g/ml) for 1 h at 42°C in 10 ml of 50% deionized formamide, 0.2% SDS, 5 × Denhardt's solution, and incubated overnight with a random-primed ( 32 P)-labeled 1 kb XbaI-HindIII fragment from the murine uPA cDNA. 34 Membranes were washed twice in 2 × SSC/0.1% SDS for 1 h at 50°C, once in 0.1 × SSC for 30 min and exposed to Kodak (Rochester, NY, USA) XAR-5 films for 1 h at RT.…”

“…This interaction involves the EGF-like domain of ATF (residues 1-46) and it exhibits species restriction: this is the case between mice and humans for which critical residues 19-32 are not conserved. [34][35][36] Preclinical data indicate that ATF-based antagonists constitute potent anticancer molecules. For example, intraperitoneal bolus injections of a human ATF-based antagonist has been shown to inhibit lung metastases of human tumor cells grown in athymic mice, with no significant effect on primary tumor growth.…”

Regulation of the plasminogen activator inhibitor-2 (PAI-2) gene in murine macrophages. Demonstration of a novel pattern of responsiveness to bacterial endotoxin

“…26]; uPA; 1.2-kb PstI fragment of murine cDNA from pDB15 [29]; murine IL-1␤ [30]; ␤-actin, a 1.8-kb chicken ␤-actin cDNA fragment in pGEM2; c-fms, p755 containing the 3.7-kb mouse c-fms cDNA, obtained from L. Rohrschneider (Fred Hutchinson Cancer Research Centre, Seattle, WA); and lysozyme, pMLC, which contains a 780-bp fragment of mouse lysozyme M cDNA, obtained from R. Renkawitz (Max Planck Institute, Munich, Germany). Blots were reprobed with a synthetic 24-bp oligonucleotide (5Ј-CAT GGT AGG CAC GGC GAC TAC CAT-3Ј) complementary to murine 18S rRNA to control for gel loading.…”

The receptor for the plasminogen activator of urokinase type is up‐regulated in transformed rat thyroid cells

“…The human uPAR is attached to the membrane via a glycosyl-phosphatidylinositol molecule Table 1 Radial "Radial ¢aseinolytie assay [25] of 4 days-conditioned medium of I-5G cells incubated with uPA for 1 h in the presence or in the absence of 10 -'~ M uPA pcptides from different species (sec Fig, 7) and treated thereafter with neutral buffer or glycine buffer, pH 3.0. bAverag¢ values, with standard deviations, from tlare¢ experiments in duplicate, Any of the three uPA peptides, '~This value was assumed as 100%. [27,28], porcine (PUPA) [29], baboon (BuPA) [30], human (HuPA) [31], and chicken (CuPA) [9] aPA growth factor-like domains are listed below; residues identical to the rat sequence are indicated with "dash~,…”