Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.     

Elimination of hydrocortisone from the medium enables tissue plasminogen activator gene expression by normal and immortalized nonmalignant human epithelial cells.

Human cervical epithelial cells transfected and immortalized with human papillomavirus type 16 DNA (HCE16/3) can be, like many other epithelial cells, normally grown in medium supplemented with epidermal growth factor, cholera toxin, hydrocortisone, insulin, transferrin, thyroid hormone and serum. We found that hydrocortisone diminished tissue plasminogen activator (tPA) production to an undetectable level. The removal of hydrocortisone increased urokinase plasminogen activator (uPA) activity within 24-48 h and tPA activity within 48-72 h, and converted the cells to a more elongated and fibroblastic phenotype. Upregulation of uPA mRNA was seen as early as at 3 h and of tPA mRNA within 48-72 h. Higher molecular weight forms (97-110 kDa) of plasminogen activators were seen in zymograms, apparently complexed with PAI-1, starting at 6 h both in the presence and absence of hydrocortisone. Immunoprecipitation with a PAI-1 monoclonal antibody confirmed that both uPA and tPA were complexed. We also studied normal diploid human bronchial epithelial cells (NHBE) and NHBE cells transformed with an adeno-12/SV40 hybrid virus (BEAS-2B). In both types of nonmalignant epithelial cells, the removal of hydrocortisone increased uPA activity. The omission of hydrocortisone increased tPA levels significantly in BEAS-2B cell cultures, and in NHBE cell cultures tPA became detectable at 72 h. No PA complexes were seen in these two cell types. We conclude that normal and immortalized nonmalignant epithelial cells produce tPA, but only if hydrocortisone is omitted in the growth medium.     

Measuring plasminogen activator inhibitor activity in plasma by two enzymatic assays

We compared two methods that measure plasminogen activator inhibitor (PAI) activity in plasma based on the ability of PAI to inhibit tissue plasminogen activator (tPA) or urokinase (uPA) in order to determine which method most accurately measures plasma PAI activity after stressors, like hemorrhage. Plasma PAI activity was significantly elevated after hemorrhage in both assays. Using standard curves derived from rhPAI-1, we found that the tPA-PAI assay was more sensitive than the uPA-PAI assay. However, we measured a 10-fold difference in PAI activity as measured between assays, suggesting that some endogenous plasma constituents (tPA, uPA, plasminogen or plasmin) may interfere with the accurate determination of PAI activity. Increasing the amount of plasma in each assay led to a progressive increase in PAI activity. However, removing either tPA or plasminogen from the tPA-PAI assay unmasked the presence of some endogenous tPA and plasminogen. Furthermore, increasing plasma volume in either assay increases measured plasma tPA, but not uPA. Finally, plasma tPA is elevated after hemorrhage, whereas plasma uPA is not. These results suggest that endogenous tPA and plasminogen may interfere with the measurement of plasma PAI activity in the tPA-PAI assay after hemorrhage or other stresses. The uPA-PAI assay does not have this confounding problem because endogenous uPA does not interfere with the assay, nor does it rise during hemorrhage.     

uPA and MMP‐2 were involved in self‐assembled network formation in a two dimensional co‐culture model of bone marrow stromal cells and endothelial cells

Two dimensional (2D) co‐cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self‐assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), matrix metalloproteinase‐2 (MMP‐2) as well tissue inhibitors of matrix metalloproteinase‐2 (TIMP‐2) were investigated in this 2D co‐culture model. We found that the expression of uPA, MMP‐2 in the co‐cultured cells was significantly higher than those in mono‐cultured cells. In opposite, PAI‐1, expressed only by HUVECs is not regulated in the co‐culture. Inhibition assays confirm that uPA played a critical role in the formation of self‐assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP‐2 prevented the formation of self‐assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP‐2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP‐2 activity, and may contribute to endothelial cell migration and the formation of this self‐assembled network observed in these 2D co‐cultured cells. J. Cell. Biochem. 114: 650–657, 2013. © 2012 Wiley Periodicals, Inc.     

人子宫内膜纤蛋白溶酶元激活因子及其抑制因子...

Two types of plasminogen activator (PAs) are present in human endometrium, and their contents vary with the different phases of menstrual cycle, i.e. high in the proliferative phase and low in the secretory phase. In the present study by immunohistochemical technique, both uPA and tPA antigens were demonstrated in the stromal and glandular cells of the endometrium. In cell culture, tPA was released only from stromal cells and uPA only from glandular cells as determined by SDS-PAGE followed by fibrin overlay technique, but PA inhibitor type-1 (PAI-1) was secreted by both stromal and glandular cells. Furthermore, secretion of PAs from endometrial cells was enhanced by adding estradiol and markedly inhibited by progesterone in a dose dependent manner, while the PAI reacted just in the opposite way. The effect of the peptide hormones, hCG, GnRH, PRL, as well as cAMP in cell culture on the secretion of PAs and PAI was similar to that of estradiol, while forskolin demonstrated definitely more stimulative effect on tPA than uPA. Taking into account of the finding of the present study, it appears that, under hormonal control, a balance between PAs and PAI in the endometrium exists. The physiological roles of the PAs and PAI in the endometrium were discussed.     

The expression and localization of urokinase-type plasminogen activator and its type 1 inhibitor are regulated by retinoic acid and fibroblast growth factor in human teratocarcinoma cells.

Human Tera 2 embryonal carcinoma cells switch gradually from rapidly growing undifferentiated cells to almost nonproliferating cells during retinoic acid (RA)-induced neuronal differentiation. This process is associated with the increased expression of type 1 plasminogen activator inhibitor (PAI 1) mRNA, and the secreted inhibitor is immobilized to the pericellular area. Furthermore, the differentiation is accompanied by a decrease in the amount of both the secreted tissue-type PA (tPA) and the mainly cell-associated urokinase-type PA (uPA) activity. In RA-differentiated cells, uPA becomes localized at the vinculin-rich cell-substratum adhesion sites. Fibroblast growth factor activity has been associated with various events during embryonal growth and with the regulation of proteolytic enzymes. A short-term treatment of the undifferentiated Tera 2 cells with basic fibroblast growth factor (bFGF) increases uPA mRNA levels and the cell-associated uPA activity, whereas the secretory tPA activity decreases. bFGF induces PAI 1 mRNA expression in the undifferentiated cells, but unlike PAI 1 protein after RA-treatment, the inhibitor does not accumulate around the cells but is released in the medium. A similar exposure to bFGF has less effect on the RA-differentiated Tera 2 cells. Under these conditions bFGF treatment leads to an increase in the amounts of PAI 1 and uPA mRNAs, but no changes in the localization of these components can be seen. Differentiation of human embryonal carcinoma cells is thus connected with an altered response to bFGF.     

人子宫内膜纤蛋白溶酶元激活因子及其抑制因子的分布与调控

人子宫内膜中存在组织型(tPA)及尿激酶型(uPA)两类纤蛋白溶酶元激活因子,其含量在增殖期高于分泌期。本文应用免疫组织化学定位证实uPA及tPA两类抗原存在于子宫内膜的腺体细胞和间质细胞中。应用SDS-PAGE分高蛋白质,继而应用纤蛋白-琼脂糖铺盖技术测得离体培养下间质细胞仅释放tPA,腺体细胞仅释放uPA,但两种细胞均分泌PA的抑制因子(PAI)。培液中加入孕酮,明显抑制PA和刺激PAI生成。雌二醇作用与孕酮相反。某些肽类激素hCG、PRL、GnRH及cAMP作用基本与雌二醇相同。但福司克林(FK)则刺激间质、腺体两种细胞产生tPA及少量uPA,抑制PAI生成。本工作表明人子宫内膜中存在PA及PAI作用相反的酶,受激素调控,其生理意义尚待进一步探讨。     

Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue-type plasminogen activator

It is well established that rat ovarian granulosa cells produce tissue plasminogen activator (tPA). The synthesis and secretion of the enzyme are induced by gonadotropins, and correlate well with the time of follicular rupture in vivo. We have found that in contrast, mouse granulosa cells produce a different form of plasminogen activator, the urokinase-type (uPA). As with tPA synthesis in the rat, uPA production by mouse granulosa cells is induced by gonadotropins, dibutyryl cAMP, and prostaglandin E2. However, dexamethasone, a drug which has no effect on tPA synthesis in rat cells inhibits uPA synthesis in the mouse. Results of these determinations made in cell culture were corroborated by examining follicular fluid, which is secreted in vivo predominantly by granulosa cells, from stimulated rat and mouse ovarian follicles. Rat follicular fluid contained only tPA, and mouse follicular fluid only uPA, indicating that in vivo, granulosa cells from the two species are secreting different enzymes. The difference in the type of plasminogen activator produced by the rat and mouse granulosa cells was confirmed at the messenger RNA level. After hormone stimulation, only tPA mRNA was present in rat cells, whereas only uPA mRNA was found in mouse cells. Furthermore, the regulation of uPA levels in mouse cells occurs via transient modulation of steady-state levels of mRNA, a pattern similar to that seen with tPA in rat cells.     

Modulation of protein expression levels and DNA methylation status of breast cancer metastasis genes by anthracycline-based chemotherapy and the demethylating agent decitabine

Epigenetic drugs are promising add‐ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline‐based chemotherapy [5‐fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA‐MB‐231 (estrogen receptor‐negative) and MCF‐7 (estrogen receptor‐positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real‐time polymerase chain reaction (PCR) (Methylight®) was employed to document the methylation status of the metastasis‐relevant urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor‐I (PAI‐1) genes. Additionally, protein expression levels of uPA and PAI‐1 were determined using enzyme‐linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel® invasion assay. Decitabine lowered the viability of MCF‐7 cells, although MDA‐MB‐231 cells were not affected. Decitabine did not augment FEC‐mediated cytotoxicity in both cell lines. In MCF‐7 cells, methylation of the uPA and PAI‐1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI‐1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF‐7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA‐MB‐231. Our results suggest differential effects of single‐dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour. Copyright © 2011 John Wiley & Sons, Ltd.     

Inhibition of human breast cancer cell (MBA-MD-231) invasion by the Ea4-peptide of rainbow trout pro-IGF-I

It was shown previously that Ea4-peptide of trout pro-IGF-I exerted mitogenic activity in non-transformed cells and inhibited colony formation in a soft agar medium of established human cancer cells. Here we report that the same peptide inhibits the invasion of human breast cancer cells (MDA-MB-231) through a matrigel membrane in a dose-dependent manner. The expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI1) genes in MDA-MB-231 cells were downregulated by treatment with rtEa4-peptide. The inhibition of expression of these genes in response to rtEa4-peptide treatment was reduced to the control level when inhibitors for c-Jun N-terminal kinase 1/2 (JNK1/2), mitogen activated protein kinase kinase 1/2 (Mek1/2), p38 mitogen activated protein kinase (p38 MAPK), phosphatidylinositol 3-kinase (PI3K), and phosphokinase C (PKC) were used. These results suggest that inhibition of invasion of MDA-MB-231 cells by rtEa4-peptide may be mediated via the suppression of uPA, tPA, and PAI1 gene activities through signal transduction pathways.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: A key event in melanoma cell invasiveness in vitro

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on Me Wo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Establishment of permanent human endothelial cells achieved by transfection with SV40 large T antigen that retain typical phenotypical and functional characteristics

Summary The plasmid pMK16 containing-SV40 replicated origin defective gene was efficiently introduced into early-passage human umbilical vein endothelial cells (HUVEC) using positively charged liposomes. The resulting cell line acquired an almost infinite lifespan, was morphologically unchanged, expressed SV40-antigen, and coexpressed von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin conversion enzyme (ACE), and endothelin converting enzyme (ECE). In addition, these are the first immortalized human endothelial cells, to our knowledge, that biosynthesized and secreted interleukins (IL-1β and IL-6) in both a constitutive and regulated fashion and endothelin-1 (ET-1), the most potent vasoactive peptide, which has been suggested to be implicated in the pathogenesis of hypertension. Interestingly enough, both of the immortalized cells and the early-passage HUVEC from which the immortalized cells were obtained biosynthesized and secreted the same levels of ET-1 suggesting full maintenance of its biosynthetic pathway including the presence of active ECE, which cleaves big endothelin-1 (big-ET-1) to ET-1 and regulation factors. Moreover, the immortalized cells retained the ability to express the functional specific amino acid Na⁺-independent system Y⁺ transporter, which mediates L-arginine transport into endothelial cells from which endothelium-derived relaxing factor (EDRF, nitric oxide) is formed via the action of nitric oxide-synthase. Obtaining these immortalized human endothelial cells without alteration of the differentiated characteristics constitutes a useful model: (a) to study ET-1 secretion, gene regulation, and human ECE, which may be an important therapeutic target in disease conditions in which ET-1 is to be implicated; (b) to study L-arginine transport, which is a key step in the formation of EDRF; (c) to study IL-1β and IL-6 secretions, and gene regulations; (d) to substitute large quantities of HUVEC; and, finally, (e) to reproduce, starting with different primary endothelial cells both from human and animal origin.     

Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line

24 established melanoma cell cultures were screened for their secretion of plasminogen activators and plasminogen activator inhibitors into the culture medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by conventional and reverse fibrin autography. Among the cell lines investigated, 22 cell lines predominantly secreting tissue type plasminogen activator (t-PA) and four cell lines additionally secreting urokinase were found. The conditioned media of two cell lines (KRFM and MJZJ) were found to contain plasminogen activator inhibitor (PAI) activity at a Mr position of approximately 50,000. The PAI of one of the two melanoma cell (MJZJ)-conditioned media found to contain PAI activity was purified to apparent homogeneity employing concanavalin A-Sepharose chromatography, gel filtration on Sephadex G-150, chromatography on Affi-Gel blue, and affinity chromatography on a Sepharose 4B immobilized monoclonal anti-t-PA IgG column. The purified melanoma PAI was found to be a single chain protein, acid stable, immunologically related to the endothelial derived PAI. In contrast to endothelial PAI, melanoma PAI presented itself in the conditioned media of the melanoma cells and in the purified preparation to an appreciable extent in its active form.     

Synthesis of Urokinase-Type Plasminogen Activator and of Type- 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.     

Urokinase and tissue type plasminogen activators in human keratinocyte culture

Using immunocytochemical and biochemical techniques, we have demonstrated that cultured human epidermal keratinocytes contain both urokinase and tissue type plasminogen activators. In subconfluent colonies the distribution of the two enzymes differed. Tissue type plasminogen activator (tPA) was distributed evenly throughout the colony, while, as we have demonstrated previously, urokinase type plasminogen activator (uPA) was preferentially localized at the migrating edges of the colony. Using zymographic analyses, both tPA and uPA activities were detected in cell extracts. Depending on the procedure used to prepare cell extracts, tPA was detected either as free enzyme or in complex with PA inhibitor type 1. PA inhibitor type 1 was deposited onto the extracellular matrix of the keratinocyte cultures and formed a complex with cell-associated tPA when cells and matrix were extracted together. The most differentiated keratinocytes in the culture, which were spontaneously shed from the culture surface, also contained both tPA and uPA. However, these spontaneously shed cells had a higher ratio of tPA:uPA than did the less differentiated cells from the same culture. In conjunction with our previous studies, these results demonstrate the complex nature of the plasminogen activator system, including enzymes and inhibitors, that is present in human keratinocytes. In addition, our data suggest that the relative amounts of uPA and tPA in epidermal cells vary with differentiation state.     

Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro.

During ovarian follicle growth, there is expansion of the basal lamina and changes in the follicular extracellular matrix (ECM) that are mediated in part by proteolytic enzyme cascades regulated by tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). One PA inhibitor, serine protease inhibitor-E2 (SERPINE2) is expressed in granulosa but not theca cells, and expression changes with follicle development. In this study, we hypothesized that PA and SERPINE2 expression/secretion by granulosa cells are regulated by FSH and growth factors. SERPINE2 mRNA and protein levels, tPA gene expression and uPA secretion were stimulated by FSH. Insulin-like growth factor-I stimulated SERPINE2 secretion and uPA activity, and decreased secreted tPA activity and gene expression. Bone morphogenetic protein-7 increased SERPINE2 secretion and expression and tPA secretion. In contrast, fibroblast growth factor-2 inhibited tPA secretion and SERPINE2 secretion and expression. Epidermal growth factor inhibited SERPINE2 secretion and expression, but increased secreted tPA activity. Estradiol and SERPINE2 secretion were highly positively correlated, but estradiol did not alter SERPINE2 expression. These data demonstrate that SERPINE2 expression and protein secretion are regulated by FSH and growth factors in non-luteinizing bovine granulosa cells. As estradiol is a known marker of follicle health, and SERPINE2 is an anti-apoptotic factor, we propose that SERPINE2 is involved in the regulation of atresia in bovine follicles.     

Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells.

Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase.     

Expression of transfected transforming growth factor alpha induces a motile fibroblast-like phenotype with extracellular matrix-degrading potential in a rat bladder carcinoma cell line.

Acquisition of cell motility is often correlated with the malignant progression of a transformed cell. To investigate some of the mechanisms involved in the development of a migratory state, we transfected the NBTII rat carcinoma cell line, which forms stationary epithelial clusters in culture, with the gene encoding human transforming growth factor alpha (TGF alpha). Expression of TGF alpha in NBTII cells resulted in cells of motile and vimentin-positive phenotype with internalized desmosomal components, analogous to the treatment of cells with exogenous TGF alpha. The clones expressed a 5.2-kb TGF alpha message and synthesized an 18-kDa form of TGF alpha. Supernatants of TGF alpha-producing clones induced the internalization of desmosomal components, the production of vimentin, and increased motility in untransfected epithelial NBTII cells, indicating that the factor produced by the clones was in a biologically active form. TGF alpha-producing clones secreted significant levels of a 95-kDa gelatinolytic metal-loproteinase, virtually absent in untransfected cell supernatants. In contrast, levels of inhibitors of metalloproteinases and of a plasminogen activator were similar in untransfected and TGF alpha-transfected NBTII cells. These results suggest that expression of TGF alpha in an epithelial tumor cell results in the development of a motile, fibroblast-like phenotype with matrix-degrading potential, which could result in a more aggressive tumor in vivo.