PubMed Health⌕ Search

Biomedical subjects

K Danø

Publications and source records attributed to K Danø.

At least 109 records · Page 6Linked to original sources
PubMed1991

Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor.

We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro-u-PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u-PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system.

Antibodies, Monoclonal↗
PubMed1991

The plasminogen activation system in human colon cancer: messenger RNA for the inhibitor PAI-1 is located in endothelial cells in the tumor stroma.

Fourteen human colon adenocarcinomas were examined by in situ hybridization for the presence of mRNA for plasminogen activator inhibitor type 1 (PAI-1). All specimens contained PAI-1 mRNA in endothelial cells of some vessels in the stroma immediately surrounding the invasive tumor glands, in granulation tissue, and in some capillaries located under the free luminal surface of carcinomatous epithelium. In addition, a limited number of stromal cells in the cancerous areas located at the periphery of newly formed capillary networks, and presumably representing sprouting endothelial cells, contained PAI-1 mRNA. Cancer cells were devoid of detectable PAI-1 mRNA in all cases. PAI-1 mRNA was not seen in three biopsies of normal colon. Together with previous findings of urokinase-type plasminogen activator and its mRNA being located in fibroblast-like cells in the tumor stroma and mRNA for the urokinase receptor in the cancer cells at invasive foci, these results indicate a complex cooperativity among several cell types in regulation of plasminogen activation in colon cancer. A possible role of PAI-1 in protecting the extracellular matrix in the tumor tissue against degradation and a role in tumor-induced angiogenesis are discussed.

Adenocarcinoma↗
PubMed1991

Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor.

The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by uPA specifically bound to its receptor on the human monocytoid cell-line U937 and demonstrate this process to have properties differing widely from those observed for uPA in solution. The solution-phase reaction was characterized by a Km of 25 microM and for the cell-associated reaction this fell 40-fold to 0.67 microM, below the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding antagonist. In contrast to intact cells, purified uPA receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937 cells) was observed to partially inhibit uPA-catalyzed Plg activation, although activity against low molecular weight substrates was retained. Therefore, the cellular binding of Plg appears to be of critical importance for the efficient activation of Plg by receptor-bound uPA. Plasmin generated in the cell-surface Plg activation system described here was also observed to be protected from its principal physiological inhibitor alpha-2-antiplasmin. Together, these data demonstrate that the cell surface constitutes the preferential site for Plg activation when uPA is bound to its specific cellular receptor, which therefore has the necessary characteristics to play an efficient role in the generation of pericellular proteolytic activity.

Antifibrinolytic Agents↗
PubMed1991

The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator.

The purified urokinase plasminogen activator receptor (u-PAR) was cleaved into two fragments by mild chymotrypsin treatment. The smaller fragment (apparent Mr 16,000) possessed the ligand-binding capability, as shown by chemical cross-linking analysis. This fragment constituted the NH2-terminal part of the intact receptor, probably including the whole sequence 1-87, and contained N-linked carbohydrate. After detergent phase separation in the Triton X-114 system, the fragment was present in the water phase where its binding activity could be demonstrated in the absence of the rest of the protein. An analysis of internal homology in the amino acid sequence of u-PAR revealed the presence of three repeats of approximately 90 residues each. The ligand-binding fragment corresponds to the first repeat, supporting that this unit is a structurally autonomous domain. Domains homologous with the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential applications in interfering with cell-surface plasmin-mediated proteolysis.

Amino Acid Sequence↗
PubMed1991

Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells.

We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand. We conclude that PMA stimulates transcription of the u-PAR gene in U937 cells, and this is responsible at least in part for the accumulation of the u-PAR mRNA and for the subsequent increase in urokinase-binding capacity.

Blotting, Northern↗
PubMed1991

Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol.

The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid analysis of u-PAR after micropurification by affinity chromatography and N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound u-PAR is efficiently released from the surface of human U937 cells by trace amounts of purified bacterial phosphatidylinositol-specific phospholipase C. This soluble form of u-PAR retains the binding specificity toward both u-PA and its amino-terminal fragment holding the receptor-binding domain. 3) Treatment of purified u-PAR with phosphatidylinositol-specific phospholipase C or mild alkali completely alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase separation and charge-shift electrophoresis. 4) Biosynthetic labeling of u-PAR was obtained with [3H]ethanolamine and myo-[3H]inositol. 5) Finally, comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.

Amino Acids↗
PubMed1991

Urokinase-receptor biosynthesis, mRNA level and gene transcription are increased by transforming growth factor beta 1 in human A549 lung carcinoma cells.

We have compared the cell-specific expression and regulation of the receptor for urokinase-type plasminogen activator (u-PAR) by transforming growth factor beta type 1 (TGF-beta 1) in 10 human cell lines derived from both normal and neoplastic tissues. The basal expression of u-PAR mRNA as well as its response to TGF-beta 1 varied strongly between different cell lines; however, five out of the 10 cell lines responded to TGF-beta 1 by an increase in the u-PAR mRNA level. Among these, A549 cells were selected for a detailed elucidation of the molecular mechanism involved in TGF-beta 1 regulation of u-PAR mRNA expression. TGF-beta 1 caused an early increase in u-PAR mRNA level, with a maximal 15-fold enhancement after 24 h of treatment. This was paralleled by an increase in u-PAR protein as detected by crosslinking studies with radiolabeled ligand, and also resulted in an increase in cell surface plasmin generation. The protein synthesis inhibitor cycloheximide also increased the level of u-PAR mRNA in a time-dependent fashion and when both cycloheximide and TGF-beta 1 were used, an additive effect was seen. Nuclear run-on experiments demonstrated only a moderate (3-fold) increase in the u-PAR gene transcription rate after exposure of the cells to TGF-beta 1 for 3 h compared with a 12-fold increase in the mRNA level. TGF-beta 1 also caused an increase of both u-PA and PAI-1 antigens, while there was no detectable effect on t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1991

Metastatic behavior of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor, and urokinase-mediated matrix degradation.

Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.

Animals↗
PubMed1991

Two alternatively spliced mouse urokinase receptor mRNAs with different histological localization in the gastrointestinal tract.

Two mouse urokinase-type plasminogen activator receptor (muPAR) cDNAs were isolated: muPAR1 is homologous to the human urokinase-type plasminogen activator receptor while muPAR2 codes for a 199 residue protein sharing the first 133 residues with muPAR1. Mouse genomic DNA sequencing indicates that the two different mRNAs arise by alternative splicing. In situ hybridization showed differential expression of the two mRNAs in mouse gastric mucosa. muPAR1 mRNA is located in luminal epithelial cells situated close to urokinase-type plasminogen activator-producing connective tissue cells of the lamina propria, pointing to plasmin generation controlled by the cooperation of different cells that may play a role in the release of gastric epithelial cells. muPAR2 mRNA is expressed in the basal epithelial cells, and the deduced protein sequence includes the receptor ligand binding domain, but omits the region involved in glycolipid-mediated membrane anchoring, suggesting that muPAR2 may code for a secreted uPA binding protein.

Amino Acid Sequence↗
PubMed1991

Complementation between urokinase-producing and receptor-producing cells in extracellular matrix degradation.

The respective roles of urokinase plasminogen activator (u-PA) and the u-PA receptor in extracellular matrix degradation was investigated. Human pro-u-PA and the human u-PA receptor were expressed independently by two different mouse LB6 cell lines. The matrix degradation capacity of these cell lines individually or in coculture was studied. Although pro-u-PA-producing cells alone degrade the matrix in the presence of plasminogen, u-PA-receptor producing cells do not. Cocultivation of a small fraction of pro-u-PA-producing cells with the receptor-producing cells increases the rate of matrix degradation at least threefold. By immunoprecipitation it was shown that cocultivation of the two cell lines increases the conversion of the inactive pro-u-PA to the active two chain u-PA. The enhancement of matrix degradation and of pro-u-PA activation requires actual binding of pro-u-PA to its receptor because it is inhibited by u-PA-receptor antagonists. The u-PA receptor must be cell associated, as binding of pro-u-PA to a receptor solubilized from the cell surface with phosphatidyl-inositol specific phospholipase C did not enhance the activation of pro-u-PA in the presence of plasminogen. The finding that activity of u-PA is enhanced when it is bound to its receptor, even when the receptor is produced by a different cell, might have important implications for the mechanisms of u-PA-induced extracellular proteolysis in vivo.

Animals↗
PubMed1991

Differential expression of urokinase-type plasminogen activator and its type-1 inhibitor during healing of mouse skin wounds.

The expression of urokinase-type plasminogen activator (u-PA) and its type-1 inhibitor (PAI-1) was examined in vivo in mouse wounds by in situ hybridization and immunohistochemistry. u-PA mRNA was present in both basal and suprabasal keratinocytes in the regenerative epithelial outgrowths at the edge of the wounds. In the same area, PAI-1 mRNA was only present in the basal keratinocytes. u-PA protein was detected in keratinocytes in several layers of the epithelial outgrowth, whereas PAI-1 protein was confined to the basal keratinocytes and to the area of the basal membrane. The two proteins and their mRNA were not detected in normal epidermis or in normal-looking epidermis adjacent to the wounds. Fibroblast-like cells and fairly large stellate cells (possibly macrophages) in the granulation tissue underneath the wound contained both the two proteins and their mRNA. The large stellate cells, showing a strong hybridization signal for PAI-1 mRNA, were especially abundant at the border between the necrotic wound and the newly formed granulation tissue. The specificity of these results was supported by the use of two different non-overlapping antisense probes, sense mRNA probes, antibody preparations preabsorbed with purified proteins, and Northern analysis of tissue extracts. The localized and regulated expression of u-PA and PAI-1 seen in this study may reflect that plasminogen activation plays a role in the migration of keratinocytes and connective tissue cells during reepithelialization and tissue remodeling in wound healing.

Animals↗
PubMed1991

Localization of urokinase-type plasminogen activator messenger RNA in the normal mouse by in situ hybridization.

The histological distribution of urokinase-type plasminogen activator (u-PA) mRNA was analyzed in normal mouse tissue by in situ hybridization with anti-sense RNA transcribed from three different subclones of a mouse u-PA cDNA. Hybridization signal was found over a distinct fibroblast-like cell in the lamina propria of the gastrointestinal tract, over proximal, distal, and collecting tubules of the kidney, and over the epithelium of the bladder, ductus deferens, and epididymis. No hybridization signal was found over cells of the lung, pancreas, liver, adrenal, pituitary, cerebrum, hypothalamus, cerebellum, sciatic nerve, and striated muscle, nor over endothelial cells in any tissue investigated. The lack of u-PA mRNA in lung tissue was confirmed by Northern analysis and is in contrast to the high amounts of u-PA protein found in this tissue.

Animals↗
PubMed1991

Localization of urokinase-type plasminogen activator in stromal cells in adenocarcinomas of the colon in humans.

Human colon adenocarcinomas and adjacent normal colon tissues were stained immunohistochemically with three different monoclonal antibodies and one preparation of polyclonal antibodies against each of the two plasminogen activators, uPA (urokinase type) and tPA (tissue type). The staining patterns seen with the respective sets of antibodies were identical. In all of 10 cases, staining for uPA in the normal colon tissue was confined to scattered fibroblastlike cells in the lamina propria. Other cells, including epithelial and endothelial cells, were uPA negative. All the tumor infiltrates contained many more uPA-positive cells than the normal tissues, but the staining was confined to fibroblastlike cells and endothelial cells in the tumor stroma, while no staining of the malignant epithelial cells was detected. Analysis for uPA by enzyme-linked immunosorbent assay (ELISA) in four cases showed an average uPA content of 0.15 ng uPA/mg protein in the normal colon tissues and 1.6 ng uPA/mg protein in the tumors. Tissue-type plasminogen activator immunoreactivity was confined to endothelial cells in both the normal colon tissue and in the colon carcinomas. These findings may indicate that colon cancer cells recruit stromal cells to produce uPA involved in degradation of the extracellular matrix during invasive growth.

Adenocarcinoma↗
PubMed1991

Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.

In this study in situ hybridization methods were used to examine biopsy samples from 13 adenocarcinomas of the colon for the presence of mRNA for the urokinase-type plasminogen activator (u-PA) and its specific cell-surface receptor (u-PAR). In all cases, u-PA mRNA was present in fibroblastlike cells in the stroma adjacent to the invasive tumor nodules. Urokinase-type plasminogen activator mRNA was not detected in the malignant cells. All specimens also contained u-PAR mRNA in cells located at the tumoral-stromal interface of invasive foci, but in contrast at least some of these cells were in all but one case identified as being of malignant origin. Stromal cells, probably tumor-infiltrating macrophages and neutrophils, also were positive in these areas. These results support the view that components of the plasminogen activation system may act to influence proteolytic events occurring at the interface between stroma and malignant cells in adenocarcinomas of the colon in humans.

Adenocarcinoma↗
PubMed1990

Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors.

Urokinase-type plasminogen activator (uPA) binds to a specific receptor on various cell types, the bound molecule retaining its enzymatic activity against plasminogen. We have now investigated whether receptor-bound uPA also retains the ability to react with and be inhibited by plasminogen activator inhibitors (PAI-1 and PAI-2). uPA bound to its receptor on human U937 monocyte-like cells was inhibited by PAI-1 (in its active form in the presence of vitronectin fragments) with an association rate constant of 4.5 x 10(6) M-1 s-1, which was 40% lower than that obtained for uPA in solution (7.9 x 10(6) M-1 s-1). The inhibition of uPA by PAI-2 was decreased to a similar extent by receptor binding, falling from 5.3 x 10(5) to 3.3 x 10(5) M-1 s-1. Stimulation of U937 cells with phorbol 12-myristate 13-acetate was accompanied by a further reduction in receptor-bound uPA inhibition by PAI-1 and PAI-2 to 1.7 x 10(6) and 1.1 x 10(5) M-1 s-1, respectively. These constants although lower than those for uPA in solution still represent rather rapid inhibition of the enzyme, and demonstrate that uPA bound to its specific cellular receptor remains available for efficient inhibition by PAI's, which may therefore play a major role in controlling cell-surface plasminogen activation and extracellular proteolytic activity.

Antibodies↗
PubMed1990

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants.

The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein to that predicted for the u-PA receptor in the same cross-linking studies; 4) the identical extent of glycosylation of the purified protein and of the u-PA receptor in crude membrane fractions, as detected after cross-linking; 5) the ability of antibodies raised against the purified protein to inhibit cellular binding of the amino-terminal fragment of u-PA.

Amino Acid Sequence↗