Human endothelial cells contain one type of plasminogen activator

133

Abstract. 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-PAmediated 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 sub-RING metastasis, tumor cells must penetrate basement membranes and interstitial tissues, when they detach from the primary tumor and intravasate into the circulation, and later when they extravasate at the site formation of the secondary tumor. This means that these tumor cells should express the right panel and adequate levels of proteolytic enzymes to degrade the extracellular matrix (15,16,24,34,49,57) . In addition, these enzymes may have a function in the process of angiogenesis when endothelial cells grow invasively into the newly formed tumor and form new blood vessels (25,38) . The serine protease plasmin is one ofthe major enzymes believed to be involved in such proteolytic processes (15,16,24,42,43) . Plasmin has a broad substrate specificity and can digest most of the components of the extracellular matrix including the basement membrane, either directly or by activation of proenzymes of metalloproteinases, like type IV collagenase or interstitial collagenase (23,39,57) . Plasmin is formed by a conversion of the zymogen plasminogen, which is regulated by plasminogen activators. Two distinct plasminogen activators The two u-PA and PAM 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.are known, the tissue-type (t-PA),' and the urokinase-type (u-PA) . The activity ofthe activators can be regulated by interactions with specific inhibitors, of which two have been described, type 1(PAI-1) and type 2 (PAI-2) (33, 53) . In addition, u-PA and its proenzyme, pro-u-PA, can be localized at cell surfaces by binding through their growth factor domain to a specific receptor (u-PAR) (2,8,12,41,51,55,56,62) .To study the role of plasminoge...

Section: Discussionmentioning

confidence: 89%

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.

Quax¹,

Muijen²,

Weening-Verhoeff³

et al. 1991

133

“…Polyclonal purified rabbit IgG against u-PA was prepared as described elsewhere (29) and detected both active u-PA and its proenzyme (31). Monoclonal anti-vinculin mouse IgGj was from Bio-Yeda (Kiryat Weizmann, Rehovot, Israel).…”

Section: Lmmunofluorescencementioning

confidence: 99%

“…For double immunofluorescent studies of u-PA and vinculin/fibronectin the cells were treated in the following sequence: rabbit anti-u-PA IgG, goat anti-rabbit IgG-TRITC, monoclonal anti-vinculin/fpg-9, sheep antimouse IgG-FITC. Immunocytochemical controls consisted of anti-u-PAIgG preabsorbed with highly purified u-PA (29), preimmune IgG from the u-PA-immunized rabbit or mouse monoclonal anti-TNP-IgG, as well as omission of any one of the steps in the staining procedure. After rinsing with PBS the coverslips were mounted in glycerol-PBS.…”

Section: Lmmunofluorescencementioning

confidence: 99%

Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts.

Pöllänen

Hedman

Nielsen

et al. 1988

297

117

Abstract.We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PAcontaining focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5 % FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion. rIESIVE interactions of cells with the extracellular matrix and their reversal are critical events for morphogenetic movements during embryonic development and for cancer cell invasion during metastasis (30, 49). The molecular structures at the sites where cultured cells contact each other and the growth substratum are thus of great interest. Focal contacts, the sites of closest cell-substratum apposition, can be visualized either by electron microscopy (1) or by interference reflection microscopy (23). Focal contacts are localized at the termini of actin-containing microfilament bundles, and contain vinculin (7, 19) and talin (6) on the cytoplasmic side, but apparently do not have fibronectin on the extracellular side (9). The so-called close contacts exhibit a greater substrate separation distance, do not contain vinculin, and probably represent a more labile type of contact (23). A third kind of substrate adhesion site termed the fibronexus (42) or extracellular matrix contact site (9) is characteristic of well-spread and stationary fibroblasts. It features a codistribution of fibronectin, its plasma membrane receptor, actin, vinculin, and alpha-actinin throughout the entire contact site and is located at central regions of the cell rather than at its margins (11,15). Fibronexuses show very close transmembrane associations of actin microfilaments and fibronectin fibrils (42,43).

“…Polyclonal purified rabbit IgG and monoclonal (clone 12) mouse antibodies against u-PA, and mouse monoclonal (clone 1) and affinity-purified polyclonal rabbit IgG antibodies against PAl-1 from the HT-1080 cell line were prepared as described elsewhere (4,34,49,50). Monoclonal antibody against the trinitrophenyl group, anti-TNP-IgG, was from the hybridoma clone designated Hy 2.15 (63).…”

Section: Immunofluorescencementioning

confidence: 99%

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells

Pöllänen¹,

Saksela²,

Salonen³

et al. 1987

296

127

Abstract. We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogenous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAIl, we stained the cells live at 0°C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10 -6 M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PAmediated focal proteolysis.