| 1. |
- Galway, A B, et al.
(författare)
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Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells : mediation by pathways independent of protein kinases-A and -C.
- 1989
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 125:1, s. 126-35
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Tidskriftsartikel (refereegranskat)abstract
- Recent reports suggest that epidermal growth factor (EGF) or related peptides may act as local hormones to regulate granulosa cell differentiation. While FSH and GnRH are known to stimulate accumulation of tissue-type plasminogen activator (tPA) mRNA in granulosa cells, studies using nonovarian cells have shown stimulation of tPA by EGF. In this study, the effect of EGF and its structural analog transforming growth factor-alpha (TGF alpha) on ovarian tPA mRNA and activity was investigated. Granulosa cells obtained from immature estrogen-treated rats were cultured with FSH or increasing doses of EGF or TGF alpha before analysis of tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique. Like FSH and GnRH, EGF and TGF alpha stimulated the secretion of tPA activity in a dose- and time-dependent manner (onset, 12 h; maximum, 48 h). Northern blot hybridization of total RNA using a rat cRNA probe for tPA showed the accumulation of a 22S species mRNA in cells treated with EGF or TGF alpha, but not with nerve growth factor, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells confirmed a time-dependent increase in tPA mRNA preceding that in enzyme activity. Cotreatment of a saturating dose of EGF with phorbol myristate acetate (PMA) or GnRH resulted in additive increases in both tPA enzyme activity and mRNA levels. In addition, pretreatment with PMA desensitized the cells to subsequent treatment with PMA or GnRH, but did not diminish EGF-induced tPA mRNA, suggesting that EGF acts through a pathway independent of protein kinase-C. Also, extracellular cAMP levels did not increase with EGF treatment in the presence or absence of a phosphodiesterase inhibitor, suggesting the lack of involvement of the protein kinase-A pathway. Suppression of protein synthesis by cycloheximide inhibited the induction of tPA mRNA by EGF, whereas similar treatment resulted in the superinduction of tPA mRNA in FSH-treated cells, suggesting that EGF and FSH do not share the same pathway. These results suggest that EGF and TGF alpha induce tPA mRNA and activity in granulosa cells through a pathway independent of protein kinases-A (FSH) and -C (GnRH and phorbol ester), providing an interesting model for future elucidation of the molecular mechanism involved in tPA gene expression.
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| 2. |
- Loskutoff, D J, et al.
(författare)
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Fibrinolytic system of cultured endothelial cells : regulation by plasminogen activator inhibitor.
- 1986
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 32:4, s. 273-80
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
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| 4. |
- Ny, Tor, et al.
(författare)
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Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor.
- 1986
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 83:18, s. 6776-80
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Tidskriftsartikel (refereegranskat)abstract
- A lambda gt11 expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the beta-migrating plasminogen activator inhibitor (beta-PAI) purified from cultured bovine aortic endothelial cells. Thirty-four positive clones were isolated after screening 7 X 10(5) phages. Three clones (lambda 1.2, lambda 3, and lambda 9.2) were randomly picked and further characterized. These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively. Escherichia coli lysogenic for lambda 9.2, but not for lambda gt11, produced a fusion protein of 180 kDa that was recognized by affinity-purified antibodies against the bovine aortic endothelial cell beta-PAI and had beta-PAI activity when analyzed by reverse fibrin autography. The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long. It has a large 3' untranslated region [1788 bp, excluding the poly(A) tail] and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5' terminus. The two clones carrying the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3' untranslated region. Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe. Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript. The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human beta-PAI. Based on this alignment, the mature human beta-PAI is 379 amino acids long and contains an NH2-terminal valine. The deduced amino acid sequence has extensive (30%) homology with alpha 1-antitrypsin and antithrombin III, indicating that the beta-PAI is a member of the serine proteinase inhibitor (serpin) superfamily.
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| 5. |
- Ny, Tor, et al.
(författare)
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Regulation of tissue-type plasminogen activator activity and messenger RNA levels by gonadotropin-releasing hormone in cultured rat granulosa cells and cumulus-oocyte complexes.
- 1987
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 262:24, s. 11790-3
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Tidskriftsartikel (refereegranskat)abstract
- Gonadotropin-releasing hormone (GnRH) acts directly on the ovary to induce ovulation in hypophysectomized proestrous rats. Because plasminogen activators (PAs) are implicated in gonadotropin-induced ovulation, we have studied the effect of GnRH on ovarian PA synthesis. GnRH induced tissue-type PA (tPA) secretion by cultured rat granulosa cells, but inhibited the secretion of urokinase-type PA. These effects were blocked by co-treatment with a GnRH antagonist, suggesting that stereospecific GnRH receptors are involved. Follicle-stimulating hormone (FSH) also induced tPA in granulosa cells but with a different time course than GnRH; the combined effect of FSH and GnRH was additive. The GnRH effect was mimicked by the calcium- and phospholipid-dependent protein kinase C activator, phorbol myristate acetate. In isolated cumulus-oocyte complexes and cumulus cells, GnRH treatment also increased tPA activity. In contrast, treatment of denuded oocytes with GnRH did not increase enzyme activity. After GnRH stimulation of the cumulus-oocyte complexes, tPA content in the denuded oocyte was elevated, suggesting that the cumulus cells mediate the action of GnRH to increase the oocyte enzyme levels. Hybridization experiments using a labeled rat tPA-specific DNA probe showed that both FSH and GnRH increased the level of tPA mRNA in cultured granulosa cells; the stimulatory effect of GnRH was blocked by the GnRH antagonist. Our results indicate that GnRH treatment increases tPA secretion by cultured granulosa cells and cumulus-oocyte complexes. The stimulation of enzyme activity in the granulosa cells is accompanied by increases in tPA mRNA levels.
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| 6. |
- Ohlsson, M, et al.
(författare)
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Hormonal regulation of tissue-type plasminogen activator messenger ribonucleic acid levels in rat granulosa cells : mechanisms of induction by follicle-stimulating hormone and gonadotropin releasing hormone.
- 1988
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Ingår i: Molecular Endocrinology. - 0888-8809 .- 1944-9917. ; 2:9, s. 854-61
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Tidskriftsartikel (refereegranskat)abstract
- FSH and GnRH both stimulate rat granulosa cells to produce tissue-type plasminogen activator (tPA). We have studied the molecular mechanisms involved in the action of these hormones by measuring tPA mRNA levels in primary cultures of rat granulosa cells. When granulosa cells were cultured in the presence of FSH or GnRH the level of tPA mRNA was increased 20- and 12-fold, respectively. The induction of tPA mRNA by FSH and GnRH was additive and the kinetics of induction differed. The effect of FSH could be mimicked by bromo-cAMP or forskolin, and was drastically enhanced by cotreatment with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. These findings are consistent with the notion that FSH mediates its effect through the protein kinase A pathway. GnRH is believed to augment phospholipid turnover in granulosa cells, leading to the activation of the protein kinase C pathway. Like GnRH, the protein kinase C activator phorbol myristate acetate also induced tPA mRNA in granulosa cells. In the presence of the protein synthesis inhibitor, cycloheximide, FSH-stimulated tPA message levels were enhanced by 30-fold, revealing superinduction of tPA mRNA levels by this pathway. In contrast the induction of tPA mRNA by GnRH was inhibited by cycloheximide indicating that the synthesis of an intermediate protein is required for the GnRH effect. Our data suggest that FSH and GnRH increase the tPA mRNA levels by two distinct pathways in cultured granulosa cells, providing a model system for studying the hormonal regulation of tPA gene expression.
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| 7. |
- Sawdey, M, et al.
(författare)
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Messenger RNA for plasminogen activator inhibitor.
- 1986
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 41:2, s. 151-60
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Tidskriftsartikel (refereegranskat)abstract
- We report here the identification and preliminary characterization of the messenger RNA coding for a Mr 50,000 plasminogen activator inhibitor (PAI) synthesized by cultured bovine aortic endothelial cells. Polyadenylated RNA was prepared from these cells and translated in a rabbit reticulocyte lysate in vitro translation system. When the 35S methionine labeled translation products were immunoprecipitated with monospecific antiserum to PAI and analyzed by SDS-PAGE and autoradiography, a single major polypeptide of Mr 40,000 was revealed. Competition experiments were performed to determine the relationship of the immunoprecipitated polypeptide to the PAI. The amount of 35S-labeled immunoprecipitate was greatly decreased by the presence of the purified PAI, consistent with the conclusion that the Mr 40,000 band represented the translation product of PAI mRNA. This mRNA migrated with a sedimentation coefficient of 22s when analyzed by sucrose gradient centrifugation. The in vitro translation assay was used to determine the relative amount of PAI mRNA in cells cultured in calf serum purchased from different vendors. The level of PAI mRNA varied by at least eight-fold depending on the serum employed, suggesting that expression of the PAI gene is subject to regulation by external factors.
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| 8. |
- Tripputi, P, et al.
(författare)
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Tissue-type plasminogen activator gene is on chromosome 8.
- 1986
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Ingår i: Cytogenetics and Cell Genetics. - 0301-0171 .- 1421-9816. ; 42:1-2, s. 24-8
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Tidskriftsartikel (refereegranskat)abstract
- Tissue plasminogen activator is one of the two plasminogen activators, both serine proteases, that catalyze the conversion of inactive plasminogen to plasmin, which then degrades the fibrin network of blood clots. By combining somatic cell genetics, in situ hybridization, and Southern blot hybridization, we localized the human tissue plasminogen activator gene to the pericentromeric region of chromosome 8.
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