Regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured rat Sertoli and Leydig cells

New data are provided to show that (i) rat Sertoli cells produce two types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), and a plasminogen activator inhibitor type-1 (PAI-1); (ii) both tPA (but not uPA) and PAI-1 secretion in the culture are modified by FSH, forskolin, dbcAMP, GnRH, PMA and growth factors (EGF and FGF), but not by hCG and androstenedione (△4); (iii) in vitro secretion of tPA and PA-PAI-1 complexes of Sertoli cells are greatly enhanced by presence of Leydig cells which produce negligible tPA but measurable PAI-1 activity;(iv) combination culture of Sertoli and Leydig cells remarkably increases FSH-induced PAI-1 activity and decreases hCG- and forskolin-induced inhibitor activity as compared with that of two cell types cultured alone. These data suggest that rat Sertoli cells, similar to ovarian granulosa cells, are capable of secreting both tPA and uPA, as well as PAI-1. The interaction of Sertoli cells and Leydig cells is essential for the cells to response to     

人组织型纤溶酶原激活剂突变体微小基因的构建

tPA基因全长约36kb,至少由13个内含子分隔为14个外显子。根据tPA的第一、二外显子的编码情况,考虑建立从第二至第六外显子序列在内的tPA微小基因。即将tPA的部分基因组序列与LAtPA cDNA的序列在第六外显子的NarI位点处相连。     

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophesis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.     

Characterization of morphogenetic and invasive abilities of human mammary epithelial cells: Correlation with variations of urokinase-type plasminogen activator activity and type-1 plasminogen activator inhibitor level

Urokinase-type plasminogen activator (uPA) and one of its inhibitors, the PAI-1, are involved in the proteolytic cascade of matrix degradation during in vivo morphogenesis or metastasis. In the present study, we have characterized the in vitro morphological behavior of human normal and malignant mammary epithelial cells and determined the levels of uPA activity and PAI-1 during these events. Two-dimensional cultures in the presence of inductive fibroblast-conditioned medium (CM) allowed migration of HBL-100 cells and MDA-MB-231 cells. Normal human mammary epithelial cells (HMEC) and MCF-7 cells failed to migrate under these conditions. The epithelial cell migration correlated with an increase in the uPA activity whereas their immobility correlated with both increases in uPA activity and PAI-1 level. In three-dimensional cultures in collagen gel, fibroblasts or fibroblast CM induced branching tubular morphogenesis to HMEC, cord-like extensions to HBL-100 cells and a greater invasiveness ability to MDA-MB-231 cells. These events correlated with an increased uPA activity. In contrast, no morphological rearrangement was observed in MCF-7 cells and this correlated with both increases in uPA activity and PAI-1 level. Altogether, these results show that the in vitro mammary epithelial behavior is under the influence of mesenchymal inductive signals and is in agreement with modifications of uPA activity and PAI-1 levels. Our culture system gives a suitable model to study the mechanisms of mammary development and metastasis and to highlight the involvement of proteases and their inhibitors in cell-cell positioning and cell-matrix reorganization.     

Intracellular localization of protein C inhibitor (PCI) and urinary plasminogen activator in renal tubular epithelial cells from humans and human PCI gene transgenic mice

Urinary plasminogen activator (uPA) is a serine protease that plays important roles in various extracellular proteolytic processes. In humans, protein C inhibitor (PCI) is known to regulate the activity of the serine proteases involved in blood coagulation, wound healing, and tumor metastasis, whereas PCI is not present in murine plasma or tissues other than the reproductive tissues. The large amount of uPA–PCI complexes found in human urine suggests that these complexes are formed in the kidneys. In the present study, we performed immunofluorescence double labeling and electron microscopic immunocytochemistry using renal tissues from humans and human PCI gene transgenic (PCI-TG) mice. In human renal tissues, PCI and uPA colocalized in the cytoplasm of renal proximal tubular epithelial cells (RPTECs), and juxtaposition of PCI and uPA immunoreactive particles was detected in the microvilli and lysosomes in the RPTECs. The intracellular distributions of PCI and uPA in the RPTECs from PCI-TG mice were similar to those observed in human RPTECs. These findings hint at the physiological roles of uPA and PCI in human kidneys, and also suggest that the PCI-TG mice will be useful for evaluating the roles of PCI in human physiological and pathological conditions.     

Interaction between plasminogen activator inhibitor type-2 and pre-mRNA processing factor 8

Plasminogen activator inhibitor type-2 (PAI-2), a mem-ber of the ovalbunin family of serine protease inhibitor (ov-serpin) super family, is a multifunctional protein which isinvolved in the regulation of fibrinolysis, invasion andmetastasis of cancer cells, and regulation of apoptosis. HeLacells transfected with PAI-2 were protected from TNF-α-induced apoptosis. Like other members of ov-serpin family,PAI-2 contains a non-conserved 33 amino acid residuesloop region between its C and D he…     

促乳素抑制培养的大鼠颗粒细胞中卵泡素介导的组织纤溶酶原激活因子的表达

本文主要是观察促乳素（ＰＲＬ）是否曩体外培养的大鼠颗粒细胞中,组织纤溶酶原激活因子（ｔＰＡ）和Ｉ型纤溶酶原激活因子抑制因子（ＰＡＩ－Ｉ）基因表达间的协调作用。我们采用了多种方法,例如ＳＤＳ－ＰＡＧＥ、免疫印迹等,来检测ＰＲＬ对ｔＰＡ和ＰＡＩ－Ｉ基因表达的作用。结果证实：（１）在离体条件下促乳素（ＰＲＬ）能刺激颗粒细胞（ＧＣ）中ＰＡＩ－Ｉ ｍＲＮＡ的合成,而ＦＳＨ无此作用。但ＦＳＨ可与ＰＲＬ协同增加     

Structure and function of human tissue-type plasminogen activator (t-PA)

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.     

Cloning and high-level expression of plasminogen activator inhibitor-1 cDNA derived from human glomerular mesangial cells

Ｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒｉｎｈｉｂｉｔｏｒ1(ＰＡＩ1)ｉｓａｓｐｅｃｉｆｉｃｐｈｙｓｉｏｌｏｇｉｃａｌｉｎｈｉｂｉｔｏｒｏｆｕｒｏｋｉｎａｓｅｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ(ｕＰＡ)ａｎｄｔｉｓｓｕｅｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ(ｔＰＡ)[1].ＣｈａｎｇｅｓｏｆＰＡＩ1ｍａｙｉｎｄｕｃｅｉｍｂａｌａｎｃｅｂｅｔｗｅｅｎｇｌｏｍｅｒｕｌａｒｅｘｔｒａｃｅｌｌｕｌａｒｍａｔｒｉｘ(ＥＣＭ)ｓｙｎｔｈｅｓｉｓａｎｄｄｅｇｒａｄａｔｉｏｎ,ｔｈｕｓｌｅａｄｉｎｇ…     

Fibrinolytic system of cultured endothelial cells: regulation by plasminogen activator inhibitor

Cultured bovine aortic endothelial cells have a relatively complex fibrinolytic system that is responsive to both the physiological state of the cell itself and to a variety of agents added to the culture medium. The fibrinolytic activity of these cells results from the production of both urokinase-type and tissue-type plasminogen activators and is regulated by an inhibitor capable of neutralizing their activities. The properties of these fibrinolytic components will be reviewed, and their respective roles in initiating and regulating the fibrinolytic activity of the cells will be summarized. A cDNA coding for the inhibitor has been isolated, and its sequence will be compared to that of other serine proteinase inhibitors.     

Inhibition of apoptosis and caspase-3 in vascular smooth muscle cells by plasminogen activator inhibitor type-1

Increased expression of plasminogen activator inhibitor type 1 (PAI-1) is associated with decreased apoptosis of neoplastic cells. We sought to determine whether PAI-1 alters apoptosis in vascular smooth muscle cells (VSMC) and, if so, by what mechanisms. A twofold increase in the expression of PAI-1 was induced in VSMC from transgenic mice with the use of the SM-22alpha gene promoter (SM22-PAI+). Cultured VSMC from SM22-PAI+ mice were more resistant to apoptosis induced by tumor necrosis factor plus phorbol myristate acetate or palmitic acid compared with VSMC from negative control littermates. Both wild type (WT) and a stable active mutant form of PAI-1 (Active) inhibited caspase-3 amidolytic activity in cell lysates while a serpin-defective mutant (Mut) PAI-1 did not. Similarly, both WT and Active PAI-1 decreased amidolytic activity of purified caspase-3, whereas Mut PAI-1 did not. WT but not Mut PAI-1 decreased the cleavage of poly-[ADP-ribose]-polymerase (PARP), the physiological substrate of caspase-3. Noncovalent physical interaction between caspase-3 and PAI-1 was demonstrable with the use of both qualitative and quantitative in vitro binding assays. High affinity binding was eliminated by mutations that block PAI-1 serpin activity. Accordingly, attenuated apoptosis resulting from elevated expression of PAI-1 by VSMC may be attributable, at least in part, to reversible inhibition of caspase-3 by active PAI-1.     

Collagen modulates gene activation of plasminogen activator system molecules

Skin extracellular matrix (ECM) molecules regulate a variety of cellular activities, including cell movement, which are central to wound healing and metastasis. Regulated cell movement is modulated by proteases and their associated molecules, including the serine proteases urinary-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) and their inhibitors (PAIs). As a result of wounding and loss of basement membrane structure, epidermal keratinocytes can become exposed to collagen. To test the hypothesis that during wounding, exposed collagen, the most abundant ECM molecule in the skin, regulates keratinocyte PA and PAI gene expression, we utilized an in vitro model in which activated keratinocytes were cultured in dishes coated with collagen or other ECM substrates. tPA, uPA, and PAI-1 mRNA and enzymatic activity were detected when activated keratinocytes attached to fibronectin, vitronectin, collagen IV, and RGD peptide. In contrast, adhesion to collagen I and collagen III completely suppressed expression of PAI-1 mRNA and protein and further increased tPA expression and activity. Similarly, keratinocyte adhesion to laminin-1 suppressed PAI-1 mRNA and protein expression and increased tPA activity. The suppressive effect of collagen I on PAI-1 gene induction was dependent on the maintenance of its native fibrillar structure. Thus, it would appear that collagen- and laminin-regulated gene expression of molecules associated with plasminogen activation provides an additional dimension in the regulation of cell movement and matrix remodeling in skin wound healing.     

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95000–135000). This t-PA form was converted to M_r-72000 t-PA by 1.5 M NH₄OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and M_r-72000 t-PA comigrated with high-molecular-weight t-PA. From the increase in M_r of t-PA or u-PA upon complex formation, the M_r of the endothelial cell component was estimated to be 50000–70000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH₄OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involed in the complex formation. It was further noted that serum-free conditioned medium of endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.     

Translocation of plasminogen activator inhibitor-1 during serum stimulated growth of mouse embryo fibroblasts

Serum-stimulated mouse embryo fibroblasts specifically secrete two proteins of molecular weights 48,000 and 26,000. The 48 kDa protein showed affinity to concanavalin A and was precipitated by antibody to plasminogen activator inhibitor. Immunoflowcytometry using anti plasminogen activator inhibitor-1 serum indicate the presence of the 48 kDa protein in quiescent cells; this protein was virtually absent in serum-stimulated cells. The presence of the plasminogen activator inhibitor-1 related protein in quiescent cells and its absence in serum-stimulated cells in combination with the observation on the absence of this protein, in the medium of quiescent cells and its presence in the medium of stimulated cells indicate that the 48 kDa protein was transferred from the cells into the medium upon serum-stimulation. The serum-mediated transfer of plasminogen activator inhibitor-1 from the cells into the medium was inhibited by actinomycin-D suggesting that the transfer process required actinomycin-D sensitive events. Treatment of pre-labelled quiescent cells with medium containing 20% fetal calf serum resulted in the gradual transfer of the labelled 48 kDa protein to the extra cellular matrix. These studies indicate that exposure of quiescent cells to fetal calf serum results in the transfer of plasminogen activator inhibitor-1 from the cells to the growth mediumvia extracellular matrix. The translocation of the protease inhibitor from the cells to the matrix and medium may enable the cellular and possibly the membrane proteases to act on growth factors or their receptors thereby initiating the mitogenic response.     

Production of plasminogen activator by synchronized normal and rous sarcoma virus-transformed chicken fibroblasts in culture

We studied intracellular activity of the plasminogen activator within the cell cycle of chemically synchronized normal and RSV-transformed chick fibrolasts in culture. Consideration has also been given to the relationship between the plasminogen activator activity and cycles of DNA synthesis or mitosis in cycling fibroblasts after viral infection. The plasminogen activator activity of the cell lysates was assayed on [¹²⁵I]fibrin-coated Petri dishes and was expressed as the radioactivity released from the plates. Normal fibroblasts produced detectable levels of plasminogen activator in the S-phase and late G₂-phase or mitosis of the cell cycle. In contrast, RSV-transformed cells produced high levels of this activator throughout the entire cell cycle although this activity fluctuated and reached a maximum in the G₂-M periods. We also found that the level of plasminogen activator activity in the transformed fibroblasts is influenced by the cycles of DNA synthesis and that cell division is required for the appearance of plasminogen activator activity in the ‘de novo’ virus-infected cultures.     

重组人糖基化尿型纤溶酶原激活剂中试产品抗原特异性的分析

尿型纤溶酶原激活剂（ｕ－ＰＡ）是新一代的基因工程溶栓药物。建立了重组人糖基化尿型纤溶酶原激活剂的中试生产工艺。中试产品的结构正确性和一致性分析是证明产品质量的重要依据,所以,对三批产品进行了抗原特异性分析。结果,ｕ－ＰＡ三批中试产品ＳＤＳ－ＰＡＧＥ显示的蛋白区带与免疫印迹显示的免疫活性区带没有批次判别说明ｕ－ＰＡ三批中试产品表现了一致的ｕ－ＰＡ的免疫原性。     

Differentiation-dependent localisation of tissue-type plasminogen activator in human bladder urothelium

Human bladder urothelium is able to secrete tissue-type plasminogen activator (tPA). The aim of our study was to analyse localisation of tPA antigen in comparison to differentiation state of cells in samples of histologically normal urothelium and non-invasive tumours of the human urinary bladder. Twenty-five samples of normal urothelium and 31 non-invasive papillary tumours from 36 patients were examined. The presence of tPA antigen was evaluated immunohistochemically. Differentiation of superficial cells was assessed by the presence of urothelial cell differentiation markers, uroplakins (UPs; immunohistochemistry) and cell's apical surface architecture (scanning electron microscopy). All tissue samples stained anti-tPA positive. In normal urothelium, the intensity of anti-tPA staining was the strongest in superficial cells, which were well-differentiated. In tumours, all cell layers stained anti-tPA positive. The intensity of anti-tPA positive reaction in the upper cell layer correlated with the percentage of anti-UP positive superficial cells. Superficial cells showed various differentiation states. The localisation of tPA antigen in human in vivo tissue is not confined to the well-differentiated superficial cells. Our results suggest a positive correlation between tPA secretion and cell differentiation.     

Expression of an antibody-targeted plasminogen activator in Escherichia coli using chemically induced stress responses

A recombinant gene coding for an antibody-targeted urokinase-type plasminogen activator was constructed for the purpose of enhancing the thrombolytic specificity of urokinase. The recombinant gene was cloned into prokaryotic expression vector pTrcHisA, and transformed into Escherichia coli strain Rosetta (DE3). Less than 4mg of the desired protein/l could be obtained in the form of inclusion bodies. Of various inducers and enhancers of stress responses, the heat-shock enhances, streptomycin, the osmotic stress inducers, d-arabinose and sucrose, and the cold-shock enhancer, tetracycline, simulated the expression of the antibody-targeted plasminogen activator by 2- 5-fold.Revisions requested 7 July 2004; Revisions received 3 September 2004     

The cellular target for the plasminogen activator,urokinase, in human fibroblasts - 66 000-dalton protein

The effects of urokinase, the plasminogen activator of human urine, on the isolated substratum-attached pericellular matrices of cultured human lung fibroblasts were studied in serum-free conditions. Pericellular matrices were prepared from dense cultures of human lung fibroblasts after labelling of the cultures with radioactive glycine. Extraction of the cultures with sodium deoxycholate and hypotonic buffer gave a reproducible pattern of polypeptides when analyzed in polyacrylamide gels. The isolated pericellular matrix was subsequently exposed to affinity chromatography-purified urokinase. Urokinase affected a 66 000-dalton protein but none of the other matrix polypeptides. The appearance of a 62 000-dalton protein that remained attached to the matrix was seen concomitantly suggesting that it was derived from the 66 000-dalton protein. The 66 000-dalton protein is the first known cellular target for urokinase.