Tissue-specific and time-coordinated hormone regulation of plasminogen-activator-inhibitor type I and tissue-type plasminogen activator in the rat ovary during gonadotropin-induced ovulation

The plasminogen-activator system provides proteolytic activity in many biological processes. The regulation of plasminogen activation may occur at many levels including the synthesis and secretion of plasminogen activators (PA) and the specific inhibition of PA activity by inhibitors. PA-inhibitor type-1 (PAI-1) is an efficient inhibitor of tissue-type PA (tPA) and urokinase-type PA (uPA) that may therefore be instrumental for the control of plasminogen activation. To investigate if coordinated regulation of PA and PA inhibitors take place in vivo in response to physiological signals, we have examined the regulation of PAI-1 and tPA in the ovary during gonadotropin-induced ovulation. We found that PAI-1, as well as tPA activity and mRNA levels, were coordinately regulated by gonadotropins in a time-dependent and cell-specific manner, such that a surge of PA-activity was obtained just prior to ovulation. Both theca-interstitial and granulosa cells synthesized PAI-1, but their maximal PAI-1 expression occurred at different times during the periovulatory period, ensuring inhibition of proteolytic activity in ovarian extra cellular compartments both before and after ovulation. The coordinated regulation of tPA and PAI-1 in the ovary may fine-tune the peak of PA activity which may be important for the regulation of the ovulatory process.     

大鼠卵巢细胞分泌纤溶酶原激活因子抑制因子（PAI—1）的...

Rat ovarian cells produce not only plasminogen activator (tPA) but also plasminogen activator inhibitor type 1 (PAI-1), and their coordinated geneexpression induced by gonadotropins are thought to be responsible for follicular rupture. In this study, it was demonstrated that (1) theca-interstitial compartment synthesizes the majority of PAI-1 activity in the ovary before ovulation, the follicular wall may therefore serve as a specific barrier to prevent the secretion of PA into the extrafollicular compartment; (2) Granulosa cells contribute only small amount of ovarian PAI-1 activity, but synthesize most of tissue-type plasminogen activator activity involved in the process leading to ovulation: (3) Since only matured cumulus-oocyte complexes secrete high level of tPA and PAI-1, both tPA and PAI-1 activity in the conditioned medium may be used as reliable markers for evaluating oocyte quality for in vitro fertilization.     

大鼠卵巢细胞分泌纤溶酶原激活因子抑制因子(PAI-1)的激素调节

哺乳动物卵巢合成两类纤溶酶原激活因子(PA),即组织型PA(tPA)和尿激酶型PA(uPA)。大鼠卵巢除主要合成tPA外,还分泌一种纤溶酶激活因子的抑制因子(PAI-1)。在促性腺激素作用下,卵巢tPA和PAI-1两种基因的协调表达是导致滤泡破裂的原因。本实验进一步证实,卵巢中的PAI-1主要由膜-间质细胞分泌,可能作为一种屏障限制颗粒细胞tPA分泌到滤泡外间质。当排卵来临时,两种细胞所分泌的tPA和PAI-1相互作用后仍发现有大量tPA活性。这可能是引起排卵的主要原因。因为成熟的卵丘细胞除分泌高量tPA外,还分泌大量PAI-1,在人工授精中两者有可能作为鉴定卵子优劣的可靠指标。     

Different mechanisms contribute to simultaneous inhibition of urokinase and tissue-type plasminogen activators by glucocorticoids in human ovarian carcinoma cells

Previous studies from our laboratory have demonstrated that OVCA 433 human ovarian carcinoma cells are glucocorticoid responsive by several criteria and contain high affinity, saturable, steroid-specific glucocorticoid receptors. These cells secrete both mammalian plasminogen activators (PAs), urokinase (uPA) and tissue-type PA (tPA). Treatment of OVCA 433 cells with 1 x 10(-7) M dexamethasone (Dex) for 4 days led to 77% and 83% reductions in the extracellular activities of uPA and tPA, respectively, released into serum-free conditioned medium during a 1-h period. Dex treatment led to a 71% decrease in the rate of extracellular uPA antigen accumulation, as determined by enzyme-linked immunosorbent assay, as well as a 73% reduction in steady state uPA mRNA levels. In contrast, Dex treatment led to only a 42% decrease in the rate of extracellular tPA antigen accumulation and a 48% decrease in tPA mRNA levels; such decreases were insufficient to account for the 83% reduction in tPA activity. Thus, while Dex-induced decreases in uPA antigen and mRNA levels accounted for all but 6% of the decrease in uPA activity, a large discrepancy existed between the magnitudes of decreased tPA activity and decreased tPA antigen and mRNA levels. OVCA 433 cells produce both PAI-1 and PAI-2, two specific PA inhibitors. Treatment of cells with 1 x 10(-7) M Dex for 4 days led to a 3.3-fold increase in the rate of extracellular PAI-1 accumulation, with little or no effect on PAI-2 accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

Plasminogen activator regulation in osteoblasts: parathyroid hormone inhibition of type-1 plasminogen activator inhibitor and its mRNA.

In order to determine the mechanism by which parathyroid hormone (PTH) stimulates plasminogen activator (PA) activity in rat osteoblasts, we investigated the effect of human PTH(1-34) [hPTH(1-34)] on the synthesis of mRNAs for tissue-type PA (tPA), urokinase-type PA (uPA), and PA inhibitor-1 (PAI-1), and on release of PA activity and PAI-1 protein in both normal rat calvarial osteoblasts and UMR 106-01 osteogenic sarcoma cells. hPTH(1-34) (0.25-25 nM) decreased PAI-1 mRNA and protein, and increased PA activity in both cell types in a dose-dependent manner with ED50 of about 1 nM for both responses. Forskolin and isobutylmethylxanthine also stimulated PA activity and decreased PAI-1 protein and mRNA in both cell types. hPTH(1-34) did not show any consistent effect on tPA and uPA mRNA in calvarial osteoblasts, but a modest (two-fold) increase of both mRNAs was observed in UMR 106-01 cells treated with 25 nM hPTH(1-34). However, when protein synthesis was inhibited with 100 microM cycloheximide, the increase of tPA and uPA mRNA by hPTH(1-34) was enhanced in UMR 106-01 cells and became evident in calvarial osteoblasts. Fibrin autography also revealed that hPTH(1-34) increases tPA and uPA activity, especially after cycloheximide treatment in UMR 106-01 cells. These results strongly suggest that PTH increases PA activity predominantly by decreasing PAI-1 protein production through a cyclic adenosine monophosphate (cAMP)-dependent mechanism in rat osteoblasts. The reduction of PAI-1 protein by PTH results in enhanced action of both tPA and uPA, and would contribute to the specific roles of these PAs in bone.     

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.     

Reciprocal regulation of tissue-type and urokinase-type plasminogen activators in the differentiation of murine preadipocyte line 3T3-L1 and the hormonal regulation of fibrinolytic factors in the mature adipocytes

Adipose tissue expresses a variety of genes including tumor necrosis factor alpha and type-1 plasminogen activator inhibitor (PAI-1); and these factors, produced by adipocytes, may be associated with the risk of coronary events in obesity. In this study, we characterized the production of fibrinolytic factors including tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PAI-1 in the differentiation of preadipocytes, and examined the hormonal regulation of these fibrinolytic factors in mature adipocytes. Mouse 3T3-L1 preadipocytes were employed as a model of adipocytes. Adipocyte differentiation was induced by insulin, dexamethasone, and 3-isobutyl-1-methyl xanthine (IBMX). alpha-Glycerophosphate dehydrogenase (GPDH) activity and glucose transporter 4 (GLUT4) mRNA, indices for adipocyte maturation, were induced on Day 4, and gradually increased. GPDH activity reached its maximum level on Day 14. The level of tPA, a major PA in preadipocytes, dramatically decreased with differentiation. On the other hand, that of uPA reciprocally increased. PAI-1 production was also dramatically induced concomitant with differentiation. In mature adipocytes, uPA production was dominant (25 microg/ml/24 h vs. 0.8 microg/ml/24 h for tPA). Total PA activity in the mature adipocytes was reduced by insulin or dexamethasone, but not by glucagon. Insulin, IBMX, and dexamethasone significantly decreased both uPA and tPA production, and increased PAI-1 production. Glucagon had no effect on the production of these fibrinolytic factors. Our results reveal that uPA is one of the markers for the differentiation of 3T3-L1 cells and that insulin, IBMX, and dexamethasone are potent regulators of the fibrinolytic activity in differentiated 3T3-L1 cells, reciprocally affecting PA and PAI-1 levels in them.     

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.     

小鼠排卵前后卵巢纤蛋白溶酶原激活因子活性的变化

给幼龄小鼠注射PMSG刺激滤泡生长,随后注射hCG以诱发排卵。在激素处理的不同时间取出卵巢,制备卵巢匀浆液或从卵巢中分离颗粒细胞和卵丘-卵母细胞复合体,并做离体培养。样品中组织型(tPA)和尿激酶型(uPA)纤蛋白溶酶原激活因子经SDS-凝胶电泳分离,用纤蛋白铺盖技术测定。实验结果表明,注射hCG 8h后15％的受试动物排卵,而卵巢匀浆液和颗粒细胞中tPA和uPA活性分别也在hCG注射后4和8h达到高峰。排卵后酶活性下降。卵丘-卵母细胞复合体主要含tPA,注射hCG 12—24h达到高峰。上述资料证明,tPA和uPA都参入小鼠排卵过程。因为排出的卵子中仍含有大量tPA,卵细胞的tPA除参与排卵外,可能对排卵后的一些生理过程也起重要作用。