PubMed Health⌕ Search

Biomedical subjects

O Saksela

Publications and source records attributed to O Saksela.

At least 55 records · Page 3Linked to original sources
PubMed1987

Transforming growth factor-beta is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts.

We have studied the mechanism of a transforming growth factor-beta (TGF-beta)-stimulated production of type-1 plasminogen activator inhibitor (PAI-1) in WI-38 human lung fibroblasts. TGF-beta causes an early increase in the PAI-1 mRNA level which reaches a maximal 50-fold enhancement after 8 h. Blocking of protein synthesis with cycloheximide causes an equally strong increase in the level of PAI-1 mRNA. Quantitative studies of the effect of TGF-beta on PAI-1 protein levels in cell extracts and culture media by using monoclonal antibodies are consistent with the effect on PAI-1 mRNA. The results suggest a primary effect of TGF-beta on PAI-1 gene transcription, and also suggest the possibility that the transcription of this gene in non-induced cells may be suppressed by a short-lived negatively regulating protein. Urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators are decreased in the culture media of TGF-beta-treated cells concomitantly with the increase in PAI-1 accumulation. These findings show that a primary and important biological effect of TGF-beta may be an overall decreased extracellular proteolytic activity, and give an insight into the molecular mechanisms underlying TGF-beta action at the genetic level.

Cell Line↗
PubMed1987

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

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 homogeneous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAI-1, we stained the cells live at 0 degree 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 PA-mediated focal proteolysis.

Cell Line↗
PubMed1987

The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells.

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor.

Animals↗
PubMed1987

Plasminogen activators during differentiation of the human kidney.

Tissue-type plasminogen-activator antigenicity was immunohistochemically localized in the developing glomerulus of human embryonic kidneys using antibodies raised against a highly purified HeLa-cell activator [43]. At the very beginning of the S-shaped-body stage of glomerular differentiation, tissue-type activator antigenicity seemed to be co-distributed with a marker of invading endothelial cells, i.e., Ulex europaeus lectin. However, during further stages of glomerular remodelling and maturation, this plasminogen activator was also localized around developing and proliferating visceral epithelial cells (podocytes). Antibodies against the urokinase-type plasminogen activator did not react with any elements of developing glomeruli; rather, they stained the proximal tubules in more mature parts of the kidney, as revealed by double immunostaining using antibodies against the brush border. The present results suggest that the tissue-type plasminogen activator plays a role in the differentiation of glomerular structures during nephron morphogenesis.

Cell Differentiation↗
PubMed1986

Identification and localization of urokinase-type plasminogen activator in human NK-cells.

We have studied the presence of plasminogen activators in large granular lymphocytes and other peripheral blood cells. After immunofluorescence staining with polyclonal antiserum against urokinase-type plasminogen activator, the majority of LGLs showed granular staining which was located in Golgiderived vesicles. LGLs were negative for tissue-type activator. The presence of only urokinase-type PA in LGLs was also confirmed by determining the molecular weight of the intracellular activator and by immunoblotting the antigen from solubilized cell preparations. Monocytes and granulocytes were more intensely stained with anti-u-PA than LGLs. In LGL/K562 cell conjugates the fluorescence was often located close to the contact area and the vesicular fluorescence polarized during conjugate formation. Inhibitors of PA and other serine proteinases are known to abolish NK-cell activity. We now show that they affect a later stage than programming for lysis in the cytotoxic action, suggesting a role for u-PA or other serine proteases in the lethal-hit stage of NK activity.

Antibody Specificity↗
PubMed1986

Local synthesis of plasminogen by the seminiferous tubules of the testis.

Plasminogen activation occurs through conversion of plasminogen to plasmin by plasminogen activators. In adult mammals, liver has been the only known site for plasminogen synthesis. Seminiferous tubules secrete plasminogen activator, but are behind a barrier that excludes the entrance of many macromolecules to this tissue. Therefore, it became of interest to study the existence of plasminogen in this system. After metabolic labeling, a 90 kDa lysine-binding polypeptide was found. This protein could be immunoblotted by anti-plasminogen antibody, and was shown to possess urokinase-dependent proteolytic activity. The findings suggest that plasminogen is synthesized by seminiferous tubules. We propose that local plasminogen synthesis may occur in tissues which are separated by specific anatomical barriers.

Affinity Labels↗
PubMed1986

Human tumor cells synthesize and secrete alpha-2-macroglobulin in vitro.

In previous studies we showed that human sarcoma and melanoma cell lines synthesize and secrete into culture medium a glycoprotein, migrating in urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 140,000. It is not detected in cultures of the corresponding normal cells. Conditioned medium of the melanoma cell line HMB-2, producing among the cell lines tested the largest amounts of this glycoprotein, has now been used as a source for purification of the protein. NH2-terminal amino-acid sequence determination of the purified glycoprotein showed that it is identical to human alpha 2-macroglobulin (alpha 2M). Rabbit antibodies raised against the glycoprotein specifically reacted in immunoblotting and immunodiffusion tests with alpha 2M present in human plasma. Likewise, these antibodies immunoprecipitated from the conditioned media of 35S-methionine-labelled melanoma and osteosarcoma cell lines the protein which had a molecular weight corresponding to alpha 2M. alpha 2M was also synthesized and secreted by 2 strains of fetal lung fibroblasts but not by fetal skin fibroblasts or adult skin fibroblasts autologous to the osteosarcoma cell line.

Cells, Cultured↗
PubMed1986

Transforming growth factor beta alters plasminogen activator activity in human skin fibroblasts.

Adult human skin fibroblasts were used as a model to study the effects of transforming growth factor beta (TGF beta) on the secreted plasminogen activator (PA) activity of cultured cells. TGF beta, at nanogram concentrations, enhanced the secretion of pro-PA from two fibroblast strains in a time- and dose-dependent manner. The induced enzymatic activity was inhibited by anti-urokinase antibodies and it co-migrated with purified urokinase in polyacrylamide gels. The secretion of PA activity was abolished when cycloheximide (0.1 microgram/ml) was added to the cultures. The activity was thus dependent on protein synthesis rather than just on direct activation of a plasminogen proactivator. TGF beta had only a slight mitogenic effect on the test cells. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin were ineffective alone in inducing PA. Insulin, on the contrary, had an inhibitory effect on the TGF beta-induced PA activity. In addition to its effects on the secretion of PA, TGF beta enhanced the production of a proteinase inhibitor by these cells. The results suggest a role for TGF beta in the regulation of PA activity and pericellular proteolysis in fibroblastic cells.

Antibodies↗
PubMed1986

Enhanced production and extracellular deposition of the endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta.

Cultured human embryonic lung fibroblasts were used as a model to study the effects of transforming growth factor-beta (TGF beta) on the plasminogen activator (PA) activity released by nontumorigenic cells into the culture medium. The cells were exposed to TGF beta under serum-free conditions, and the changes in PA activity and protein metabolism were analyzed by caseinolysis-in-agar assays, zymography, and polypeptide analysis. Treatment of the cells with TGF beta caused a significant decrease in the PA activity of the culture medium as analyzed by the caseinolysis-in-agar assays. The quantitatively most prominent effect of TGF beta on confluent cultures of cells was the induction of an Mr 47,000 protein, as detected by metabolic labeling. The Mr 47,000 protein was a PA inhibitor as judged by reverse zymography. It was antigenically related to a PA inhibitor secreted by HT-1080 tumor cells as demonstrated with monoclonal antibodies. The induced Mr 47,000 inhibitor was deposited into the growth substratum of the cells, as detected by metabolic labeling, immunoblotting analysis, and reverse zymography assays of extracellular matrix preparations. TGF beta also decreased the amounts of urokinase-type and tissue-type PAs accumulated in the conditioned medium, as detected by zymography. Epidermal growth factor antagonized the inhibitory effects of TGF beta by enhancing the amounts of the PAs. These results indicate that growth factors modulate the proteolytic balance of cultured cells by altering the amounts of PAs and their inhibitors.

Cell Line↗
PubMed1986

Testicular plasminogen activators during postnatal development in the rat.

In the seminiferous epithelium, Sertoli cells secrete plasminogen activator (PA) under regulation of follicle stimulating hormone, cyclic AMP and neighbouring spermatogenic cells. Recent observations suggest that preleptotene spermatocytes upon their release from the basement membrane of the seminiferous tubule are important regulators of PA secretion. To study further the role of PA's in the seminiferous tubules, we have analyzed the endogenous levels and secretion rates of PA at various ages during postnatal development, and performed biochemical analyses of the types of PA in the testis and spent media from seminiferous tubular cultures. Cyclic secretion of PA started at the age of 28 days, and from 40 days onwards, the high secretion rates were localized in stages VII and VIII of the cycle of the seminiferous epithelium. The secreted PA is most obviously of the urokinase type; both urokinase-type and tissue-type PA-like activities were found in seminiferous tubular homogenates. The increase in testicular PA levels concomitant to the onset of meiosis in the epithelium was due to the urokinase-type PA-like activity.

Aging↗
PubMed1985

Plasminogen and tissue-type plasminogen activator bind to immobilized fibronectin.

Fibronectin immobilized onto polystyrene surface was found to bind plasminogen and tissue-type plasminogen activator (t-PA) but only slightly the urokinase type as determined using mono- and polyclonal antibodies against the activators. Of the defined fibronectin fragments tested, the Mr 120,000-140,000 fragment was found to bind both plasminogen and t-PA. Proteolytically modified plasminogen (Lys-plasminogen) bound considerably better than the native form (Glu-plasminogen). Experiments with 125I-plasminogen yielded Kd = 9.1 X 10(-8) M for the binding to immobilized fibronectin. The partially or completely inactive single-chain form of t-PA (pro-t-PA) bound considerably better than the activated two-chain form. Lysine at greater than 3 mM inhibited the binding of plasminogen. The interaction was independent of calcium ions. CaCl2 (greater than 0.5 mM) and NaCl (greater than 0.2 M) inhibited the binding of pro-t-PA and of t-PA. Fibronectin-bound t-PA retained its ability to activate plasminogen. The observed interactions may operate in directional proteolysis localizing plasminogen and plasminogen activator to degrade fibronectin-containing extracellular matrix including fibrin clots.

Epitopes↗
PubMed1985

Urokinase-type plasminogen activator and its inhibitor secreted by cultured human monocyte-macrophages.

Human blood monocytes in culture differentiate to macrophagelike cells within 1 week. Coinciding with this morphological transition the cells started releasing increasing amounts of the serine proteinase plasminogen activator (PA; Mr 56,000) of the urokinase (u-PA) type and the proteinase inhibitor alpha-2-macroglobulin (alpha 2M). Unlike the cell-associated PA activity, which was also readily detected in fresh monocytes, the activity secreted into the serum-free culture medium could be measured only after treatment of the samples with sodium dodecyl sulphate. Heat or acid treatment of the medium was not sufficient to reveal the PA activity, suggesting that, apart from alpha 2M, another PA-inhibiting substance was present in the culture medium. The inhibitor (Mr 65,000) was found to be synthesized by macrophages and specifically inhibited u-PA activity but not tissue-type PA (t-PA) or plasmin activity. Dexamethasone decreased the secretion of PA by differentiated macrophages without affecting the production of alpha 2M or the PA inhibitor. Dexamethasone also inhibited the morphological differentiation of the cells when added to the monocyte-phase cells.

Cell Adhesion↗
PubMed1985

Regulation of plasminogen activator activity in human fibroblastic cells by fibrosarcoma cell-derived factors.

Low-molecular-weight protein factors (Mr 8,000 to 18,000) from serum-free conditioned medium of human fibrosarcoma (8387) cells reversibly enhanced the secretion of proteinase-inhibitory activity by cultured normal human skin fibroblasts. This inhibitory activity could be absorbed by immobilized plasminogen activator (PA) of urokinase type but not by heparin, and it was sensitive to treatment with sodium dodecyl sulfate. The secretion of a heparin-binding Mr 60,000 proteinase inhibitor, resembling protease nexin, was also detected. Early passages of adult skin fibroblasts do not contain or secrete PA. When cell types secreting this enzyme were tested, the fibrosarcoma-derived factors decreased the PA secretion detectable after sodium dodecyl sulfate treatment in all conditioned media of normal and malignant fibroblastic cells examined, including the 8387 cell line itself. However, no effects on the secretion of PA by normal or malignant cells of epithelioid origin or by melanoma cells were seen. A similar preparation from human epidermoid carcinoma (A431)-conditioned medium did not affect the PA activity or secretion of proteinase inhibitors from fibroblastic cells. The ability of sarcoma cells to modulate the production of PA inhibitors is a novel characteristic in the regulation of cellular proteolysis.

Cells, Cultured↗
PubMed1984

Plasminogen activators, activation inhibitors and alpha 2-macroglobulin produced by cultured normal and malignant human cells.

In the present paper we have characterized the plasminogen activators (PA) synthesized by 25 different human cell lines. Technically easy methods were adopted for concentration and immunological characterization of the activators even in the presence of PA inhibitors. Most cell lines produced u-PA (mol. wt 55,000), melanoma and HeLa cells t-PA (mol. wt 66,000) and two carcinoma cell lines and normal skin fibroblasts produced no detectable PA. The classical 125I-fibrin method was compared to a caseinolytic assay and some of the discrepancies between results obtained with the two methods were shown to be due to cell-derived NaDodSO4-sensitive proteinase inhibitors in culture media. Additionally, synthesis and uptake by the cells of the wide-spectrum proteinase inhibitor alpha-2-macroglobulin ( alpha 2M ) were studied by radioimmunoassay and immunofluorescence. No production of alpha 2M could be measured in any of the malignant cell lines. In normal cells no correlation existed between the production of alpha 2M and the observed inhibition of PA activity, which indicates that other proteinase inhibitors are produced by the cells.

Cells, Cultured↗
PubMed1984

Presence of alpha-2-macroglobulin in normal but not in malignant cervical epithelium.

alpha-2-Macroglobulin (alpha 2M) was demonstrated in normal and mildly dysplastic cervical epithelium using immunohistochemical methods. In contrast, staining for alpha 2M diminished gradually with advancing epithelial dysplasia and was totally negative in truly neoplastic lesions. No in vitro synthesis could be detected in cultured ectocervical cells or in a corresponding malignant cell line (HeLa). However, alpha 2M was taken up from added human serum by cultured normal ectocervical cells, whereas HeLa cells remained negative also under these conditions. These and previous results suggest that normal cells may have the capacity to synthesize or endocytose alpha 2M, thus possibly regulating their pericellular proteolysis, while these functions may be defective in malignantly transformed cells.

Cervix Uteri↗