Hormonal regulation of membrane phenotype

Incubation of rat hepatoma cells (HTC) in tissue culture with glucocorticoids alters several membrane properties characteristic of transformed cells, without affecting the growth rate of these cells. Variant cell lines resistant to dexamethasone inhibition of plasminogen activator production have been isolated using an agar-fibrin overlay technique to detect plasminogen activator production by individual colonies of HTC cells. The resistance to dexamethasone is not secondary to abnormal or absent glucocorticoid receptors, but due to a lesion in a later step in hormone action specific for plasminogen activator. These variants should prove useful for the study of the mechanism of steroid action as well as for the analysis of the role of proteases in the hormonal regulation of membrane function.     

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.     

Properties of protein kinase and adenylate cyclase-deficient variants of a macrophage-like cell line.

Stable variants of the macrophage-like cell line J774.2, defective in adenylate cyclase and protein kinase activities, were selected by cloning cells resistant to the growth-inhibitory effect of cholera toxin and 8-bromo-adenosine 3':5' cyclic monophosphoric acid (8 Br-cAMP), respectively. These variants were analyzed for their ability to respond to cyclic AMP-mediated enhancement of phagocytosis and cyclic AMP-mediated inhibition of plasminogen activator secretion and growtn. The adenylate cyclase variants were unaffected by cholera toxin but were sensitive to 8 Br-cAMP-mediated inhibition of plasminogen activator secretion and growth. One of these variants exhibited a defect in phagocytosis that could be corrected by 8 Br-cAMP. The protein kinase variants exhibited normal basal phagocytosis that could not be stimulated by either 8 Br-cAMP or cholera toxin; they were also insensitive to cyclic AMP-mediated inhibition of plasminogen activator secretion and growth. The studies demonstrate that the three effects of cyclic AMP in J774.2--inhibition of growth and plasminogen activator secretion, and enhancement of basal Fc-mediated phagocytosis--are mediated by a cyclic AMP-dependent portein kinase. The results support the usefulness of variants in cyclic nucleotide metabolism in understanding the regulation of differentiated cell function by cyclic AMP.     

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.     

Dexamethasone regulates the program of secretory glycoprotein synthesis in hepatoma tissue culture cells

The secretory glycoproteins synthesized by hepatoma tissue culture (HTC) cells were resolved by two-dimensional polyacrylamide gel electrophoresis of media from cells that were grown in the presence of [(3)H]fucose. These cells synthesize and secrete a complex set of fucose-containing glycoproteins. These secretory glycoproteins are distinct from those glycoproteins present in the plasma membrane of HTC cells. Incubation of HTC cells with dexamethasone has a pronounced effect on the quality and quantity (denoted here as the program) of secretory protein synthesis, as assayed by the short-term incorporation of labeled mannose, fucose, or methionine. The synthesis of two mannose- and fucose- containing glycoprotein series, one of 50,000 mol wt and a more heterogeneous series with mol wt of 35,000-50,000, is increased to a high level by the hormone; conversely, the synthesis of other secretory proteins, particularly one with mol wt of 70,000, is decreased or stopped completely. The synthesis of some major secretory proteins is not affected by the hormone. Dexamethasone has less of an effect on the composition of either total cell membrane glycoprotein or plasma membrane glycoprotein. But there is a decrease in the synthesis of a major membrane glycoprotein series with mol wt of 140,000. These effects of dexamethasone are relatively specific to HTC cells. Neither Reuber H-35 cells nor primary cultures of rat hepatocytes show the same response to the steroid. Two variant HTC cell lines, which were selected for their resistance to dexamethasone inhibition of extracellular plasminogen activator activity, respond only partially to the steroid-induced regulation of the secretory and membrane glycoproteins.     

Biochemical and molecular characterization of the glucocorticoid receptor of lymphosarcoma P1798 variants

Three phenotypically distinct isolates from lymphosarcoma P1798 have been compared with respect to properties of the glucocorticoid receptor. Wild type P1798 cells express functional receptors and glucocorticoid treatment of such cells causes cytolysis in vivo. Wild type cells do not undergo cytolysis in culture. Rather, such cells exhibit reversible inhibition of proliferation in the presence of dexamethasone. Two variant populations were selected from this background. One was selected for the ability to form tumors in mice receiving pharmacological doses of glucocorticoids. Cells from such tumors are resistant to the cytolytic effects of glucocorticoids in vivo, but are sensitive to the antiproliferative effects of the hormone in culture. Variants were also selected based upon their ability to proliferate in the presence of dexamethasone in culture. These variants were resistant to glucocorticoid-mediated cytolysis in vivo. Wild type P1798 cells express approximately 20,000 high affinity dexamethasone-binding sites per cell. Dexamethasone-mesylate labeling and immunoblotting experiments indicate that hormone binding is due to a polypeptide of Mr 90-100 K. This polypeptide is encoded in an mRNA species that resolved as a single entity of approximately 7000 nucleotides. Variants selected for resistance to cytolysis in vivo are indistinguishable in any of these respects from wild type cells. The receptors are fully functional, as evidenced by their ability to precipitate growth arrest of dexamethasone-treated cultures. Variants selected for resistance in culture harbor a receptor mutation. They express fewer than 500 dexamethasone-binding sites per cell. Such variants contain neither detectable dexamethasone-mesylate-binding protein nor any protein that is recognized by a receptor antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease nexin

When the plasminogen activator urokinase was radioiodinated and incubated at 40 ng/ml in medium conditioned by human foreskin (HF) cells, within 30 min over 80% of the added plasminogen activator was complexed to cell-released protease nexin (PN). The urokinase complexed to PN had little if any activity. Incubation of purified PN with urokinase confirmed that PN is an inhibitor of this plasminogen activator. However, a widely used plasminogen-dependent fibrinolysis assay for plasminogen activator indicated that abundant endogenous plasminogen activator activity co-existed with PN in HF cell-conditioned medium. The source of this activity was electrophoretically and immunologically indistinguishable from urokinase. Furthermore, gel exclusion chromatography showed that about 90% of the urokinase antigen detected in conditioned medium had a molecular weight similar to that of free active urokinase. These paradoxical findings are resolved by evidence that this "PN-resistant urokinase-like" plasminogen activator is actually urokinase proenzyme that is activated by plasmin or conditions in the fibrinolysis assay for plasminogen activator. It is shown that the activated form of HF cell plasminogen activator is sensitive to inhibition by PN. PN may thus be an important component in the cellular regulation of endogenous plasminogen activator activity.     

Dexamethasone inhibition of tissue-type plasminogen activator (tPA) activity: paradoxical induction of both tPA antigen and plasminogen activator inhibitor

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity and reveals the presence of a specific PA inhibitor (PAI-1). To determine whether the hormonal inhibition of PA activity reflects a decrease in the amount of PA or an increased amount of the inhibitor, or both, we have assayed PA and PAI-1 immunologically. HTC PA was determined to be entirely of the tissue type (tPA), and both free and complexed antigen was quantified by a RIA using rabbit antirat tPA, with rat insulinoma tPA as tracer and standard. PAI-1 was quantified by a Western blot assay using rabbit anti-HTC PAI-1 antibody and purified HTC PAI-1 as standard. Under conditions in which dexamethasone inhibited PA activity by 90%, there was no decrease in the cellular content of tPA antigen. Paradoxically, dexamethasone increased tPA antigen approximately 1.5-fold. Under these same conditions, dexamethasone increased PAI-1 antigen 4- to 5-fold. We conclude that the glucocorticoid inhibition of tPA activity in HTC cells is not secondary to a decrease in the amount of tPA but is secondary to the induction of a specific PA inhibitor.     

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.     

Specific stimulation by estradiol of tissue-type plasminogen activator production in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor cells.

The hormonal regulation of two plasminogen activators, tissue-type plasminogen activator (t-PA) and urokinase (u-PA), was studied both in 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma and in DMBA-induced rat mammary dysplasia. t-PA activity in DMBA-mammary carcinoma was decreased markedly by oophorectomy and recovered upon estradiol administration to reach the maximum level at 12 hr. In contrast to its effect on DMBA-mammary carcinoma, estradiol had no effect on t-PA activity in DMBA-mammary dysplasia. Furthermore, DMBA-mammary carcinoma cells in primary culture displayed similar estrogen-dependency in production of t-PA, while t-PA production in DMBA-mammary dysplasia cells was not under the control of estradiol in vitro. Moreover, estrogen-stimulated production of u-PA activity was not observed in DMBA-mammary carcinoma cells or DMBA-mammary dysplasia cells both in vivo and in vitro. Taken together, these results suggest that estrogen stimulates the production of t-PA but not u-PA and that this estrogen dependency of t-PA is limited to malignant DMBA-mammary tumor cells.     

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.     

Modulation of cell-associated plasminogen activator activity by cocultivation of a stem cell and its tumorigenic descendant.

The effect of the presence of one cell type on the plasminogen activator activity of another cell type was studied. The cell types, AC and D, were isolated from a rat neuroblastoma (I. Imada and N. Sueoka, Dev. Biol. 66:97-108, 1978). AC cells are stem cells capable of multipotential differentiation in vitro and have little or no cell-associated plasminogen activator activity. D cells are tumorigenic and have high levels of cell-associated plasminogen activator activity. When AC cells were cocultivated with D cells, the plasminogen activator activity of the D cells was dramatically inhibited. The presence of as few as 1,250 AC cells inhibited 70% of the plasminogen activator activity of 20,000 D cells, as determined by a highly quantitative assay. The amount of inhibition by AC cells was proportional to the number of AC cells present. At increasing numbers of AC cells and a constant number of D cells, the Vmax for the activation of plasminogen proportionately decreased and the Km remained constant, implying that AC cells did not alter the structure or concentration of plasminogen. Inhibition was not mediated by a soluble inhibitor secreted by AC cells. Rather, attachment of AC cells adjacent to D cells, i.e., cell-to-cell contact, seemed to be required for inhibition. The substratum-attached material of AC cells, that which remained on the microwell surface after removal of AC cells with EDTA, inhibited D cell plasminogen activator activity. If plasminogen activator activity is involved in metastasis, then regulation of the plasminogen activator activity of one cell type by another cell type may be involved in determining which cells in a tumor can metastasize and where secondary tumors can arise.     

Mouse L cells expressing human prourokinase-type plasminogen activator: effects on extracellular matrix degradation and invasion

A cosmid (cos pUK0322) harboring the complete human urokinase-type plasminogen activator (u-PA) gene and Geneticin resistance as a selectable marker was isolated from a human genomic library and characterized. After transfection of cos pUK0322 into mouse L cells and selection, several plasminogen activator (PA)-expressing clones were obtained and one (LuPA) was chosen for additional study. The PA expressed was identical to human pro-u-PA in enzymatic, electrophoretic, and antigenic properties. The expression of PA was stable over 50 population doublings. The regulation of the transfected gene was studied by treatment of the cells with various hormones and other effectors. Expression of PA activity was inhibited fivefold by dexamethasone and stimulated two- to threefold by agonists of the adenylate cyclase dependent pathway of signal transduction, such as dibutyryl cyclic AMP and cholera and pertussis toxins. The modulation of PA activity was associated with corresponding changes in mRNA steady-state levels. The phenotypic changes associated with pro-u-PA expression were analyzed in vitro by degradation of 3H-labeled extracellular matrix (ECM), invasion of a matrigel basement membrane analogue, and by light and electron microscopy. LuPA cells and reference HT-1080 fibrosarcoma cells, in contrast to control Lneo cells transfected with the neomycin resistance gene, degraded the ECM and invaded the matrigel basement membrane. Matrix degradation correlated with the modulation of pro-u-PA gene expression as it was inhibited by dexamethasone and promoted by dibutyryl cyclic AMP. Inhibition of PA or plasmin using anti-u-PA IgG or aprotinin prevented ECM degradation and invasion. These results demonstrate that u-PA expression alone is sufficient to confer to a cell an experimental invasive phenotype.     

K562 human erythroleukemia cell variants resistant to growth inhibition by butyrate have deficient histone acetylation

K562 is an established human erythroleukemia cell line, inducible for hemoglobin synthesis by a variety of compounds including n-butyrate. To elucidate the role of butyrate-induced histone acetylation in the regulation of gene expression in K562 cells, we isolated 20 variants resistant to the growth inhibitory effect of butyrate. Four variants having different degrees of resistance were selected for detailed study. All four were found to be resistant to the hemoglobin-inducing effect of butyrate, suggesting that the two aspects of butyrate response, restriction of growth and induction of hemoglobin synthesis, are coupled. Further, after (5 days) culture with butyrate, two of the four variants exhibit less acetylation of H3 and H4 histones than does the butyrate-treated parent. Analysis of histone deacetylases from the variants indicated that each variant was distinct and that butyrate resistance may be accounted for by decreased affinity of the variant enzymes for butyrate, increased affinity of the enzymes for acetylated histone, or both. The fact that variants selected for resistance to growth inhibition by butyrate are also deficient in butyrate-induced hemoglobin synthesis and have abnormal histone deacetylase activity argues for butyrate inducing K562 cells to synthesize hemoglobin and restrict growth via histone acetylation.     

Plasminogen activator activity in differentiating leukemia cells

Plasminogen activator (PA) activity of human promyelocytic leukemia cell line HL-60 was assayed by following the conversion of plasminogen to plasmin and the plasmin-mediated hydrolysis of 14C-labeled globin. When HL-60 cells were induced to differentiate into macrophages by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cell-associated PA activity and secretion of PA into the conditioned medium increased profoundly. PA activity increased earlier and as a result of lower concentrations of TPA than the ability of the cells to adhere. Exposure to 10(-6)M dexamethasone did not prevent TPA-induced adherence and produced a slight inhibition of cellular PA activity. These findings imply that TPA-induced differentiation of HL-60 cells to macrophage-like cells is associated with induction of PA activity.     

Decreased fibrinolytic activity in porcine-to-primate cardiac xenotransplantation.

BACKGROUND: One major barrier to successful xenotransplantation is acute vascular rejection, a process pathologically characterized by microvascular thrombosis and diffuse fibrin deposition in transplant blood vessels. This pathologic picture may result from a disturbance in the coagulant or fibrinolytic pathways that regulate normal vascular patency. This study evaluated the regulation of fibrinolytic activity defined by tissue plasminogen activator and plasminogen activator inhibitor-1 as it may exist in the setting of acute vascular rejection. MATERIALS AND METHODS, RESULTS: Serial biopsies from cardiac xenotransplants evaluated by immunofluorescence microscopy demonstrated progressive decreases in tissue plasminogen activator and increases in plasminogen activator inhibitor-1. In vitro studies measuring fibrinolytic activity of cell culture medium from porcine aortic endothelial cells stimulated with human serum or autologous porcine serum revealed that human serum triggered as much as 93% increase in antifibrinolytic activity. CONCLUSIONS: These findings demonstrate that porcine vascular endothelial cells change toward an antifibrinolytic state following stimulation with human xenoreactive antibodies and complement. The shift is at least partly explained by an increased ratio of plasminogen activator inhibitor-1 to tissue plasminogen activator, and is at least in part mediated by the activation of complement. This increased antifibrinolytic activity may contribute to the thrombotic diathesis seen in acute vascular rejection in pig-to-primate xenografts.     

Genetic analysis of dexamethasone resistance in L cells by somatic cell hybridization

Stable dexamethasone resistant and receptor-containing (R+) variants of L cells have been characterized by somatic cell hybridization. Neither of the variants had a clearly dominant phenotype in hybrids with dexamethasone-sensitive fibroblast lines, i.e. the resistance of the variants was not due to transdominant factors. Somatic cell hybrids formed between one of the R+-resistant clones and an independent resistant fibroblast cell line showed complementation--the hybrid clones were as sensitive to the steroid as the sensitive parental lines. Complementation, however, disappeared after continued culture of the clones. The return of the dexamethasone-sensitive phenotype was not always linked with similar changes in the responsiveness to another steroid, e.g. progesterone. Our clones can be considered to be resistant variants, designated death-less (d-), where the cells are defective in a non-receptor component involved in the hormone response. The fact that complementation can occur indicates the existence of at least two such steps in the pathway.     

Plasminogen activator in the rat ovary. Production and gonadotropin regulation of the enzyme in granulosa and thecal cells

The production of plasminogen activator by ovarian granulosa cells has been previously reported to be temporally correlated with ovulation in the rat and to be under hormonal control of gonadotropins. We have examined the type of plasminogen activator produced by granulosa cells and also investigated other ovarian cell types for synthesis of this enzyme. Using antibodies specific for tissue-type or urokinase-type plasminogen activator, we have found that granulosa cells produce exclusively the tissue-type enzyme. However, in cultures of whole follicles isolated from the ovary, there is primarily synthesis of urokinase-type plasminogen activator. Examination of other isolated ovarian cell types has demonstrated that thecal cells secrete the urokinase-type plasminogen activator and that the production of this enzyme is also regulated by gonadotropins and temporally correlated with ovulation. These results suggest that ovulation requires both types of plasminogen activator and that the neighboring granulosa and thecal cells cooperate to ensure rupture of the follicle wall and unimpeded passage of the ovum into the oviduct.