Hormonal regulation of plasminogen activator in rat hepatoma cells

Plasminogen activators are membrane-associated, arginine-specific serine proteases which convert the inactive plasma zymogen plasminogen to plasmin, an active, broad-spectrum serine protease. Plasmin, the major fibrinolytic enzyme in blood, also participates in a number of physiologic functions involving protein processing and tissue remodelling, and may play an important role in tumor invasion and metastasis. In HTC rat hepatoma cells in tissue culture, glucocorticoids rapidly decrease plasminogen activator (PA) activity. We have shown that this decrease is mediated by induction of a soluble inhibitor of PA activity rather than modulation of the amount of PA. The hormonally-induced inhibitor is a cellular product which specifically inhibits PA but not plasmin. We have isolated variant lines of HTC cells which are selectively resistant to the glucocorticoid inhibition of PA but retain other glucocorticoid responses. These variants lack the hormonally-induced inhibitor; PA from these variants is fully sensitive to inhibition by inhibitor from steroid-treated wild-type cells. Cyclic nucleotides dramatically stimulate PA activity in HTC cells in a time- and concentration-dependent manner. Paradoxically, glucocorticoids further enhance this stimulation. Thus glucocorticoids exert two separate and opposite effects on PA activity. The availability of glucocorticoid-resistant variant cell lines, together with the unique regulatory interactions of steroids and cyclic nucleotides, make HTC cells a useful experimental system in which to study the multihormonal regulation of plasminogen activator.     

Hormonal regulation of tissue plasminogen activator secretion and mRNA levels in rat granulosa cells

Granulosa cells from immature rats produce tissue plasminogen activator (tPA) in response to follicle stimulating hormone (FSH) or luteinizing hormone (LH) both in vitro and in vivo. We have used the in vitro system to investigate the level at which the hormonal induction of tPA is regulated. Within 12 h following FSH addition, a dramatic but transient increase in tPA secretion occurs for by 24 h secretion returns to basal levels. This pattern of enzyme induction is similar with LH, but the onset of the increase is delayed. When steady-state tPA mRNA levels are examined after hormone treatment, the results mirror those obtained if one measures enzyme activity; a large increase in tPA mRNA followed by a decrease to basal levels is observed with both hormones, and the lag in induction by LH is also apparent. These results demonstrate that the regulation of tPA activity by gonadotropins occurs at the level of the steady-state concentration of the mRNA. In the presence of cycloheximide, the induction of tPA mRNA by FSH or LH is not greatly affected, indicating that this phase of the response to gonadotropins does not require the synthesis of new protein. However, the decrease in tPA mRNA levels observed 24 h after FSH treatment is affected by cycloheximide, in that the drug delays the reduction in mRNA levels seen with hormone alone.     

Hormonal stimulation alters the type of plasminogen activator produced by Sertoli cells

Primary cultures of immature rat Sertoli cells, maintained in serum-free medium, secrete two types of plasminogen activator (PA). When cultured under basal conditions, the preparations predominantly produce PA having a relative molecular weight (Mr) of 45,000 to 48,000. This PA activity is inactivated by antiserum against urokinase-type PA. When Sertoli cells are stimulated by follicle-stimulating hormone (FSH) or by dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), PA secretion is increased. The PA produced under these conditions has an Mr of 70,000, and is inactivated by antiserum against tissue-type PA but not by antiserum against urokinase-type PA. We conclude that, under basal conditions, Sertoli cells primarily secrete PA having the characteristics of urokinase-like PA (mu PA), and that Sertoli cells stimulated by FSH or by dbcAMP predominantly produce PA having the properties of tissue-type PA (tPA). Segments of adult rat seminiferous tubules, at defined stages of the cycle of the seminiferous epithelium, also produce and secrete two types of PA into the medium when maintained in organ culture. Segments at all stages examined release primarily mu PA in preparations cultured under basal conditions. In contrast, segments cultured in the presence of FSH synthesize larger amounts of PA, predominantly of the tPA type. An additional protease, which is independent of plasminogen, is secreted by tubule segments stimulated by FSH. The activity of this novel protease is not detectable in cultures maintained under basal conditions. We discuss the data in relation to the possible role of proteases in the restructuring of the seminiferous tubule during spermatogenesis.     

Hormonal and acid-base regulation of phosphoenolpyruvate carboxykinase mRNA levels in rat kidney

Metabolic acidosis (6 days NH4Cl) causes a fourfold increase in the relative abundance of mRNA encoding phosphoenolpyruvate carboxykinase in rat kidney. Streptozotocin-diabetes (6 days) also results in an increased abundance of the mRNA but this increase can be prevented if the acidosis associated with bicarbonate is corrected by treatment with bicarbonate. The results confirm that renal phosphoenolpyruvate carboxykinase is regulated primarily by changes in acid-base status and that this control is at a pretranslational step. Isolated kidney tubules in short-term incubation have been used to identify which agents regulate levels of phosphoenolpyruvate carboxykinase mRNA. The relative abundance of the mRNA was increased by glucocorticoids and hormones which act via cAMP, or by cAMP analogues directly, but was not affected by hormones acting via Ca2+. Neither incubation at pH 7.1 nor the presence of serum from acidotic rats had any effect on the level of phosphoenolpyruvate carboxykinase mRNA. It is concluded that acidosis, glucocorticoids, and cAMP independently regulate expression of renal phosphoenolpyruvate carboxykinase.     

Retinoid modulation of plasminogen activator production in rat Sertoli cells

Tissue type (t) and urokinase type (u) plasminogen activators (PAs) have been shown to be secreted by Sertoli cells in the seminiferous tubules in a cyclic fashion and to be dependent upon FSH stimulation or upon the presence of adjacent spermatogenic cells. In the present study we have analyzed the production of PAs by retinoid-treated rat Sertoli cells. In addition, because retinoids modulate the response of Sertoli cells to FSH either potentiating or antagonizing its action, we have investigated a possible modulation of FSH-stimulated PA production. Under basal conditions, Sertoli cells, isolated from prepubertal rats, secrete predominantly uPA. A significant dose-dependent inhibition of uPA activity was observed after treatment with retinol, while no significant effect was detected upon tPA secretion. When Sertoli cells were cultured in the presence of 0.25 microM retinol, a significant inhibition of uPA activity was evident after 16 h of treatment and reached approximately 80% after 48 h of treatment. The analysis of the mRNA levels revealed that retinol induces an inhibition of the steady-state levels of uPA mRNA without affecting those of tPA. Moreover, retinol affected uPA mRNA levels by increasing mRNA turnover. The effect of retinoids on Sertoli cells isolated from older animals was less evident, possibly due to the reduced production of uPA with the increase of age of the donor animals. Our results on the effect of retinoids upon Sertoli cell uPA production reinforce the importance of retinoids in the control of postnatal testis development.     

Patterns of plasminogen activator production in cultured normal embryonic cells

Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor.     

Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.     

Hormonal regulation of sodium-dependent phosphate transport in opossum kidney cells

There are multiple regulators of renal proximal tubule sodium-dependent phosphate (Na(+)-Pi) transport, including 1,25-dihydroxyvitamin D (1,25-Vit. D), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), and arachidonic acid (AA) and/or its metabolites. The purpose of our studies was to determine whether the effect of these factors on Pi transport is synergistic or antagonistic. The control solution or the substances were added independently or coincidentally to opossum kidney (OK) cells before incubation for 4 h. 1,25-Vit. D (10(-8) M) had no significant effect on Pi transport ( upward arrow6.8%; p = 0.8). PTH (10(-7) M) significantly inhibited Pi transport by 39.6% (p < 0.0001). IGF-1 (10(-8) M) stimulated Pi transport by 19.6% (p < 0.0001). The AA metabolite 20-HETE (10(-7) M) had no significant impact on Pi transport ( downward arrow6.4; p = 0.3). The combined effect of 1,25-Vit. D and PTH was no different from PTH alone (p = 0.2). Likewise, addition of either 1,25-Vit. D or 20-HETE to IGF-1 failed to affect the magnitude of the increase on Pi transport induced by IGF-1 alone (p = 0.4, p = 0.6, respectively). The combination of 20-HETE and PTH was not different from that observed with PTH alone (p = 0.9). We conclude that in OK cells, PTH inhibits whereas IGF-1 stimulates Pi transport into OK cells. The effects of each of these hormones are independent and unaffected by either 1,25-Vit. D or 20-HETE.     

Hormonal control of urokinase plasminogen activator secretion by sheep ovarian surface epithelial cells

Secretion of urokinase plasminogen activator (uPA) by ovarian surface epithelium (OSE) adjacent to the preovulatory ovine follicle has been implicated in apical tissue degradation and follicular rupture. In vitro experiments were designed to test the hypothesis that uPA release by OSE is under direct hormonal control. Epithelial cells were isolated from the ovarian surface of sheep using a polytetrafluorethylene scraper designed to dislodge adherent cells from culture flasks. Amidolytic cleavage of a uPA-specific chromogen (carbobenzoxy-L-gamma-glutamyl [alpha-ot-but]-glycyl-arginine-p-nitroanilide monoacetate) was used as a measure of enzymatic bioactivity in OSE-conditioned incubation media. Secretion of uPA by OSE suspensions from proestrous ewes was stimulated by exposure (2 h) to a preovulatory surge-like concentration of LH. OSE cells obtained during the luteal phase or anestrus were not responsive to LH. Baseline rates of uPA secretion and expression of estradiol receptors (in situ immunofluorescence detection) were not affected by reproductive status. Induction of uPA secretion by anestrous OSE was attained after priming (6 h) with estradiol-17beta; responsiveness was attributed to gonadotropin receptor (ligand binding) up-regulation. Monolayers of OSE established on polyethylene membranes secreted uPA predominately in a basal (i.e., toward the substratum) direction. We suggest that OSE in juxtaposition with the (hyperemic) wall of the preovulatory follicle is perfused by surge levels of LH, invoking uPA release into underlying ovarian tissues.     

Tissue plasminogen activator mRNA in murine tissues

The urokinase-type and tissue-type plasminogen activators are the two enzymes found in mammals, which specifically convert the zymogen plasminogen to plasmin. Using cDNA probes, we have assayed for the presence of the two types of plasminogen activator mRNAs in murine tissues. We demonstrate that tissue-type plasminogen activator mRNA can be detected in a wide variety of tissues. In contrast, the accumulation of urokinase-type plasminogen activator mRNA is observed in only a few of the tissues analyzed. Using an S1 nuclease assay, we demonstrate that the tPA mRNA detected contains the complete sequences encoding the non-protease finger, growth-factor and kringle domains.     

Calcium regulation of tissue plasminogen activator and prostaglandin biosynthesis in HeLa cells

Phorbol 12-myristate 13-acetate, 1-20 nM, induced the synthesis in HeLa cells of a 65 200 Mr tissue-type plasminogen activator, and of prostaglandin E2. Omission of Ca2+ from the incubation medium inhibited the induction of plasminogen activator synthesis by 40-60% and abolished the induction of prostaglandin E2 synthesis. Maximal plasminogen activator synthesis could be maintained at extracellular Ca2+ concentrations of approx. 0.1 mM, while maximal prostaglandin synthesis required at least 0.45-0.9 mM Ca2+. The induction of each factor was inhibited by 10-100 microM 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular C2+ mobilization. Prostaglandin synthesis, but not plasminogen activator synthesis, was also inhibited by 10-100 microM verapamil and nifedipine, which inhibit intracellular Ca2+ uptake via the so-called 'slow-channels' and by 0.5-10 microM trifluoperazine, an inhibitor of calmodulin. Neither plasminogen activator synthesis nor prostaglandin synthesis were stimulated by 5-50 microM 1-oleoyl-2-acetylglycerol or 1-250 microM 1,2-dioctanoylglycerol, alone and in combination with 50 nM-1 microM ionophore A23187. These results indicate that the synthesis of plasminogen activator and prostaglandins in HeLa cells is Ca2+-dependent, and that the Ca2+ requirements for each process are not identical. Thus, Ca2+ regulation of the production of tissue plasminogen activator and prostaglandin E2 occurs at multiple points in their biosynthetic pathways.     

Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575–582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.     

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.     

Tumorigenicity of herpesvirus-transformed cells correlates with production of plasminogen activator.

Our studies first demonstrated that established hamster cell lines transformed in vitro by herpesviruses activate plasminogen more effectively than normal hamster fibroblasts. This ability is probably due to increased levels of the enzyme plasminogen activator (PA). In the studies described here, the 333-8-9 cell line, originally transformed by herpes simplex virus type 2 strain 333, was used to derive subclonal lines that maintained stable PA phenotypes over the course of long in vitro passage. We were interested in correlating tumor formation by the subclones with their fibrinolytic capacity. Cells were, therefore, single-cell subcloned twice, and resulting cultures were tested for ability to activate plasminogen in vitro. PA activity was then quantitated by [125I]fibrin lysis assay, and high- and low-activity subclones were isolated; these retained high- or low-activity phenotypes. Syngeneic newborn hamsters were inoculated with these subclones and observed for the appearance of palpable tumors. A strong correlation between enzyme activity and tumor formation was observed in four separate trials; animals receiving high-PA subclones developed tumors more rapidly than those inoculated with the parental cell line. Tumors were also excised from test animals, and the cell lines established from the tumors were tested in vitro at different passages for their ability to activate plasminogen. These tumor cells were then reinoculated into syngeneic animals to confirm the tumorigenicity of cell lines with high fibrinolytic activity. In these experiments, the positive correlation between PA production and tumorigenicity was confirmed.     

Endotoxin induction of plasminogen activator and plasminogen activator inhibitor type 1 mRNA in rat tissues in vivo

The tissue-specific distribution of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA) and their inhibitor type 1 (PAI-1) was analyzed at mRNA level in five major rat organ tissues. t-PA mRNA was detected in lung, kidney, heart, and liver. u-PA mRNA was detected in kidney and lung. Presence of PA mRNA correlated with the detection of PA activity in extracts of these tissues. PAI-1 mRNA was detected predominantly in heart and lung. Although PAI activity could not be measured directly in tissue extracts, the presence of PAI-1 mRNA correlated with the occurrence of PA.PAI complex in fibrin autography of tissue extracts. Endotoxin injection caused a very large increase in plasma PAI activity. This increase correlated with a marked increase in PAI-1 mRNA in nearly all tissues studied. The increase in PAI-1 mRNA is most pronounced in lung and liver. Endotoxin injection also caused an increased level of t-PA mRNA in heart and kidney, and an increased u-PA mRNA level in kidney. mRNA analysis of freshly isolated and separated subfractionated liver cells showed that the marked increase in PAI-1 mRNA in the liver after endotoxin injection may be due mainly to a strong increase of PAI-1 mRNA in the liver endothelial cells.     

Loss of glucocorticoid regulation of plasminogen activator activity in anucleate rat hepatoma cells

Glucocorticoids decrease the plasminogen activator activity of rat hepatoma cells through production of an inhibitor. We have examined the dexamethasone regulation of plasminogen activator in anucleate rat hepatoma cells to investigate the role of the nucleus in the steroid regulation of this membrane-associated phenomenon. Dexamethasone did not affect either the intra- or extra-cellular plasminogen activator activity of the anucleate cells, and did not induce production of an inhibitor of plasminogen activator. Therefore, glucocorticoid regulation of plasminogen activator activity requires the presence of an intact nucleus.     

Thyrotropin regulation of plasminogen activator activity in primary cultures of ovine thyroid cells

In primary cultures of ovine thyroid cells, a high level of plasminogen activator (PA) activity was detected in the culture media. This level is much higher than in primary cultures of Sertoli cells, granulosa cells, and pituitary cells. PA activity increased with time in culture and was regulated by TSH and insulin. Activity gel analysis of the culture media revealed a major band of 43,000 daltons and a minor one of 70,000 daltons, suggesting the presence of both of the urokinase-type and the tissue-type PA in the media.