Biomedical subjects

O Ritvos

Publications and source records attributed to O Ritvos.

53 records · Page 3Linked to original sources

PubMed1993

Transforming growth factor beta 2 in epithelial differentiation of developing teeth and odontogenic tumors.

Dysregulation of TGF beta 2, a modulator of cell growth and differentiation, can result in uncontrolled growth and tumor formation. Our comparative studies on the expression of TGF beta 2 mRNA and protein indicate that TGF beta 2 may primarily be a regulator of epithelial differentiation during tooth development (between 13 and 20 gestational wk) and tumorigenesis of odontogenic neoplasms. A paracrine mode of action for TGF beta 2 in early human tooth germ (cap/early bell stage) is suggested by location of mRNA in the mesenchyme surrounding the tooth germ, whereas protein is found in the epithelial dental lamina and enamel organ. During the late bell stage, TGF beta 2 gene expression shifted from the mesenchyme to the odontogenic epithelium and was colocalized with protein, suggesting an autocrine role for the terminal differentiation of ameloblasts. In odontogenic tumors of epithelial origin (ameloblastomas) and epithelial-ectomesencymal origin (ameloblastic fibromas), TGF beta 2 mRNA was mostly located in the mesenchymal tumor component and protein in the epithelial tumor component. Odontogenic ectomesenchymal tumors (myxomas) were not associated with TGF beta 2 mRNA and protein expression. The results imply that TGF beta 2 may play an important role in epithelial-mesenchymal interactions in human tooth morphogenesis and development of odontogenic tumors.

Adolescent↗

PubMed1992

Activin A/erythroid differentiation factor is induced during human monocyte activation.

Activin A/erythroid differentiation factor (EDF), a dimeric polypeptide hormone composed of two beta A subunits, regulates growth and erythroid differentiation of human hematopoietic progenitor and erythroleukemia cells. We have identified activated human peripheral blood monocytes as a natural source of activin A/EDF. In these cells, lipopolysaccharide (LPS) induced rapidly the expression of the beta A subunit mRNAs through protein kinase C-dependent transcriptional regulation. The beta A subunit mRNA expression was also increased by 1,25-dihydroxyvitamin D3, an inducer of macrophage maturation of monocytes. Western analysis with an anti-beta A antibody and an erythroid differentiation bioassay confirmed that the conditioned media of LPS-activated monocytes contained the activin A/EDF protein. We suggest that monocyte/macrophage-derived activin A/EDF may not only modulate hematopoiesis but may also act as a mediator molecule in the diverse physiologic and pathogenetic events in which these cells are involved.

Activins↗

PubMed1992

Regulation of the cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNAs in cultured choriocarcinoma cells.

Cytochrome P-450scc (P-450scc) catalyzes the cholesterol side-chain cleavage reaction, a rate-limiting enzymatic step for progesterone synthesis in trophoblastic and other steroidogenic cells. Adrenodoxin is the iron/sulfur protein donating electrons to P-450scc during this reaction. We examined the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12-O-tetradecanoylphorbol acetate (TPA), a phorbol ester protein kinase C activator, on the levels of mRNAs encoding P-450scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450scc and adrenodoxin mRNA levels about 3-fold and 1.5-fold above that of control, respectively. Epidermal growth factor (EGF) was found to weakly induce P-450scc mRNA accumulation with a maximal 20% stimulation above basal levels. The effects of CT and TPA were apparently additive on both mRNAs. The protein synthesis inhibitor cycloheximide diminished basal, CT-, TPA-, and EGF-stimulated P-450scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-I) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450scc and adrenodoxin mRNAs is mainly controlled by the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway but their stimulation by TPA- and EGF-induced signals may also play a weaker synergistic role; (2) the protein synthesis inhibitor cycloheximide inhibits basal, CT-, TPA- and EGF-stimulated P-450scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.

Adrenodoxin↗

PubMed1992

Regulation of aromatase cytochrome P-450 and 17 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid levels in choriocarcinoma cells.

In human placenta the enzyme complex aromatase catalyzes the conversion of androgens to estrogens and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) mediates the reversible interconversion of, e.g. estrone to estradiol. We studied the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12-O-tetradecanoyl phorbol 13-acetate (TPA), a phorbol ester protein kinase C activator, on the levels of messenger (m) RNAs encoding aromatase cytochome P-450 (P-450AROM) and 17 beta-HSD in cultured JEG-3 choriocarcinoma cells. With the use of oligonucleotide probes designed according to known complementary DNA sequences, hybridizable mRNA transcripts of 3.0, 2.4, and 1.6 kilobases for P-450AROM were found in Northern blot analysis of JEG-3 cell RNA. A single 1.4-kilobase transcript was detected for 17 beta-HSD. Time-dependent increases in P-450AROM mRNA levels in JEG-3 cells were observed for both CT and TPA with maximal effects at 24-48 h. CT and TPA increased P-450AROM mRNA levels in a concentration-dependent manner. The maximal effects, about 4.8-fold and 3.3-fold stimulations above basal levels, were obtained with 10 ng/ml of CT and 100 ng/ml of TPA, respectively. The effects of CT and TPA were additive. CT induced 17 beta-HSD mRNA levels in a time- and concentration-dependent manner and its maximal effect of 10.1-fold above basal levels was obtained within a similar time and concentration-dependence as for P-450AROM mRNA. TPA itself had no clear effect but it approximately doubled the effect of CT on 17 beta-HSD mRNA expression. Inhibition of protein synthesis by cycloheximide decreased basal, CT and TPA stimulated P-450AROM mRNA levels but increased the expression of 17 beta-HSD mRNA. This result is consistent with the hypothesis that induction of P-450AROM gene expression is mediated by a labile protein regulator resembling to most other steroidogenic P-450 enzymes, whereas 17 beta-HSD as a non-P450 enzyme appears to be controlled in a different manner. The present results suggest that: 1) induction of P-450AROM mRNA may at least partly be responsible for our previously reported increases in the rate of conversion of androgens to estrogens by CT and TPA in JEG-3 cells; 2) 17 beta-HSD mRNA expression is mainly controlled through a cAMP-dependent mechanism in contrast to the multifactorial control of P-450AROM mRNA; and 3) protein synthesis inhibition by cycloheximide has opposite effects on the mRNA levels of these two key enzymes in placental estrogen metabolism.

17-Hydroxysteroid Dehydrogenases↗

PubMed1991

Adenosine 3',5'-monophosphate and phorbol ester induce transforming growth factor-beta 1 messenger ribonucleic acid levels in choriocarcinoma cells.

Transforming growth factor-beta 1 (TGF-beta 1), a regulator of growth and differentiation of many cell types, has previously been purified from the human placenta, and the messenger (m) RNA is abundantly expressed there. We found that the approximate 2.5-kilobase TGF-beta 1 mRNA is expressed in JEG-3 human choriocarcinoma cells, which have been widely used as a model system for studying the regulation of trophoblast hormone secretion. Cholera toxin (CT) elevates the cellular levels of the second messenger cAMP and increases the secretion of CG and steroids in these cells, thus being a potent inducer of trophoblast-differentiated functions. We show that CT also stimulates TGF-beta 1 mRNA levels in JEG-3 cells in a concentration- and time-dependent manner as studied by Northern and dot blotting. The maximal effect (about 5-fold increase above basal levels) occurs within 12-48 h of induction with a CT concentration of 1.0 ng/ml. The cell-permeable cAMP-analog 8-bromo-cAMP stimulates the accumulation of TGF-beta 1 mRNA in JEG-3 cells as well. Furthermore, this cAMP analog also induces TGF-beta 1 mRNA levels in normal cultured term placental cytotrophoblasts. 12-O-Tetradecanoyl phorbol-13-acetate, an active phorbol ester protein kinase C regulator and inducer of TGF-beta 1 mRNA in many cells, increases TGF-beta 1 mRNA accumulation in JEG-3 cells with a similar time course as cAMP analogs but to a lesser extent. Human HT-1080 fibrosarcoma and A-549 lung carcinoma cells exhibit up-regulation of TGF-beta 1 mRNA in response to TGF-beta 1 itself, but we show that activation of the cAMP-dependent pathway does not affect TGF-beta 1 mRNA levels in these cells. Cycloheximide, an inhibitor of protein synthesis, prevents the effect of CT and 12-O-tetradecanoyl phorbol-13-acetate on TGF-beta 1 mRNA expression in JEG-3 cells, suggesting that a protein mediator may be involved in the transduction of their effects. Our finding of a cAMP-dependent induction pathway for TGF-beta 1 mRNA expression in JEG-3 cells provides a new mechanism for the regulation of the synthesis of this ubiquitous growth and differentiation factor and suggests that TGF-beta 1 may have a role in trophoblast differentiation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Hormonally regulated inhibin gene expression in human fetal and adult adrenals.

Inhibin subunit expression has recently been shown to occur in rat and sheep adrenals. We now show the presence of inhibin subunit mRNAs in human fetal and adult adrenal tissue specimens and cultured adrenocortical cells. Northern blot analysis revealed that inhibin alpha-subunit gene is as strongly expressed in fetal adrenals as in fetal testes, whereas adult adrenals expressed alpha-subunit mRNA to a lesser extent. beta A-Subunit mRNA was detectable in placenta, and beta B mRNA was found in testes. With reverse transcription-polymerase chain reaction analysis all inhibin subunit mRNAs (alpha, beta A, and beta B) could be found in fetal adrenal samples. In cultured fetal and adult adrenal cells ACTH and dibutyryl cAMP increased inhibin alpha-subunit mRNA 3- to 4-fold. beta A mRNA was spontaneously induced in cultured adrenal cells. 12-O-Tetradecanoyl phorbol-13-acetate, a protein kinase-C regulator, increased beta A mRNA levels 9.6- and 3.3-fold in fetal and adult adrenal cultures, respectively. 12-O-Tetradecanoyl phorbol-13-acetate treatment abolished ACTH-induced alpha-subunit mRNA accumulation in both fetal and adult cultures. Our results show that inhibin genes are expressed in human fetal adrenals and testes during the second trimester of gestation. Adult adrenals also express inhibin genes, although to a lesser extent than fetal adrenals. Both cAMP- and protein kinase-C-dependent pathways regulate inhibin subunit gene expression in adrenocortical cells. These findings suggest that inhibins/activins are produced locally in human adrenals, where they could function as paracrine or autocrine regulators of adrenal growth and steroidogenesis.

Adrenal Cortex↗

PubMed1989

Human granulosa cells synthesize low molecular weight insulin-like growth factor-binding protein.

Human follicular fluid contains insulin-like growth factor I (IGF-I) and its low molecular weight binding protein (IGF-BP). We studied the synthesis of IGF-BP by the granulosa cells obtained after ovarian hyperstimulation for in vitro fertilization. The granulosa cells were cultured for 72 hours in Ham's F-10 medium supplemented with 10% fetal calf serum (FCS). Samples of the culture medium were collected every 24 hours. The IGF-BP concentration in culture medium increased from 1.2 to 2.1 micrograms/l at 48 h and to 3.3 micrograms/l at 72 h. De novo synthesis of IGF-BP was shown by incorporation of labeled methionine into immunoreactive IGF-BP, as detected by SDS polyacrylamide electrophoresis (PAGE) and fluorography. Our results demonstrate synthesis of IGF-BP in the human ovary.

Cells, Cultured↗

PubMed1989

Regulation of insulin-like growth factor-binding protein-1 production in human granulosa-luteal cells.

Human follicular fluid contains the insulin-like growth factor-binding protein (IGFBP-1) synthesized by ovarian granulosa cells. We studied the regulation of IGFBP-1 production by the granulosa-luteal cells from the hyperstimulated follicles of patients attending an in vitro fertilization program. The cells were first allowed to attach and recover from the hyperstimulation for 2 days. Then, a protein kinase-C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA), and adenylate cyclase activators, gonadotropins, FSH, hCG, cholera toxin, or prostaglandin E2 (PGE2) were added to the cells. The gonadotropins failed to increase IGFBP-1 production, whereas it was enhanced by TPA and to a lesser extent by cholera toxin and PGE2. The maximal response to TPA occurred at the concentration of 1.0 ng/mL, and the enhancing effect of TPA was detected at 24 h. PGE2 stimulated IGFBP-1 production; the lowest effective concentration was 10(-8) mol/L. The mean highest response was 4.3-fold and occurred at the PGE2 concentration of 10(-5) mol/L. The effect of PGE2 on IGFBP-1 production became detectable at 24 h, and it continued to increase up to 72 h. PGE2 also increased granulosa-luteal cell progesterone production in a dose- and time-dependent manner. Incorporation of [35S]methionine into immunoreactive IGFBP-1, as detected by sodium dodecyl sulfate-polyacrylamide electrophoresis and fluorography, was also increased by TPA. This suggests that TPA accelerated the synthesis of IGFBP-1. Our results indicate that the production of IGFBP-1 by human granulosa-luteal cells can be regulated both via protein kinase-C- and adenylate cyclase-dependent pathways.

Cells, Cultured↗

PubMed1988

Monoclonal antibodies to the 27-34K insulin-like growth factor binding protein.

Monoclonal antibodies were prepared against the 27-34K insulin-like growth factor (IGF)-binding protein purified from human placenta/decidua and designated placental protein 12 (PP12). Four different antibodies were characterized. Each recognized the major band at 32K on immunoblots of the purified PP12 preparation and amniotic fluid. In liquid phase RIA, IGF-I did not affect the binding of [125I] PP12 to one antibody (Mab 6303), it slightly increased the binding to two antibodies (Mab 6301 and 6304), and it slightly decreased the binding to one antibody (Mab 6302). All antibodies immunoprecipitated the cross-linked PP12-[125I] IGF-I complex, but Mab 6302 considerably less effectively than the others. Preincubation of PP12 with Mab 6302 completely inhibited the binding of [125I] IGF-I to PP12, whereas preincubation with Mab 6303 had no effect, and Mab 6301 as well as Mab 6304 increased it. These results suggest that Mab 6302 binds to an epitope at or near to the IGF-binding site, whereas the other antibodies react at other sites of the PP12 molecule. Conformational changes in PP12 probably account for the IGF-I-induced increase in the binding of Mabs 6301 and 6304 to [125I] PP12, and vice versa, for Mabs 6301- and 6304-induced increase in the binding of [125I] IGF-I to PP12.

Antibodies, Monoclonal↗

PubMed1988

Differential regulation of hCG and progesterone secretion by cholera toxin and phorbol ester in human cytotrophoblasts.

The secretion of hCG and progesterone (P) by human cytotrophoblasts was studied in response to cholera toxin (CT), which activates adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C stimulator. During a 24 h incubation CT and TPA increased hCG and P secretion in a concentration-dependent manner. At maximal effective concentrations, CT (1.0 ng/ml), TPA (10 ng/ml) and CT plus TPA stimulated hCG production by 5.7-, 2.7- and 10.0-fold, respectively, and P production by 1.8-, 1.8- and 2.0-fold, respectively. Time-course studies indicated that these effects became detectable after 12 h and increased up to 48 h of incubation. During a 24 h culture TPA potentiated CT-induced cAMP formation by 1.4-fold indicating that its effects on hCG production may be, at least partly, mediated through cAMP. In conclusion, CT and TPA are potent stimulators of human cytotrophoblast hCG and P production. Simultaneous stimulation by these agents results in potentiation of hCG production whereas P production remains at the same level as with CT or TPA alone. The results suggest that the endocrine activity of human cytotrophoblasts is under multihormonal control and hCG and P secretion are differentially regulated.

Cells, Cultured↗

PubMed1988

Regulation of the production of placental protein 5 by human endometrial stromal cells; the role of prostaglandins E2 and F2 alpha.

Cultured human endometrial stromal cells were found to release placental protein 5 (PP5), a glycoprotein with properties of a serine protease inhibitor. Progesterone had no effect on PP5 release, but cholera toxin and 12-O-tetradecanoylphorbol 13-acetate stimulated PP5 release in a time- and concentration-dependent fashion. Prostaglandin E2 (PGE2) caused a parallel increase in cAMP and PP5 release in a time- and concentration-dependent fashion. The lowest PGE2 concentration which increased cAMP and PP5 release was 1 X 10(-9) M. Maximal increase in cAMP (42-fold) and PP5 (25-fold) release was obtained by 10(-5) M PGE2. Stimulation of cAMP by PGE2 was detectable at 15 min and was followed by an increased PP5 release at 24 h. The concentrations of prostaglandin F2 alpha (PGF2 alpha) which stimulated cAMP and PP5 release were pharmacological suggesting that this effect is nonspecific. The results indicate that the activation of cAMP- and protein kinase C-dependent pathways in endometrial stromal cells increases the production of PP5. PGE2 could be one of the physiological ligands employing the cAMP-dependent pathway in endometrial stromal cells.

Cells, Cultured↗

PubMed1988

Modulation of steroidogenesis in choriocarcinoma cells by cholera toxin, phorbol ester, epidermal growth factor and insulin-like growth factor I.

The effects of cholera toxin (CT), which stimulates adenylate cyclase, 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on progesterone (P) and estradiol (E2) secretion by human choriocarcinoma JEG-3 cells were studied. During a 48 h incubation, CT, TPA and EGF stimulated P production in a concentration-dependent manner, whereas IGF-I was without effect. CT (1.0 ng/ml), TPA (10 ng/ml) and EGF (10 ng/ml) stimulated P production maximally 4.3-, 3.3- and 2.3-fold over basal, respectively. When added together with CT, TPA and EGF stimulated P production 10.0- and 5.0-fold over basal production showing that the effects of CT plus TPA were more than additive but those of CT plus EGF less than additive. Time-course studies indicated that the effects were detectable at 12 h, and continued to increase up to 48 h. The conversion of added dehydroepiandrosterone sulfate (DHEAS) to E2 was stimulated by CT and TPA and inhibited by IGF-I in a concentration-dependent manner. By contrast, EGF had no effect. The maximal responses in E2 production were 3.2- and 2.0-fold over unstimulated cells by CT (1.0 ng/ml) and TPA (10 ng/ml), respectively. When both agents were added together, their effects on E2 production were additive with 5.5-fold increase over unstimulated cells. IGF-I (30 ng/ml) inhibited maximally basal and CT-stimulated E2 production by 33% and 42%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholera Toxin↗

PubMed1988

Characterization of functional type I insulin-like growth factor receptors from human choriocarcinoma cells.

Specific receptors for insulin-like growth factor I (IGF-I) on cultured human choriocarcinoma cells (JEG-3 and BeWo) were characterized. The binding of 125I-labeled recombinant (Thr59)IGF-I to the cells was reversible and time, temperature, and pH dependent. Steady state of binding occurred after 16 h at 4 C, pH 7.4. Natural human IGF-I (hIGF-I), hIGF-II, recombinant (N-Met)IGF-I, rat multiplication-stimulating activity, and insulin were 200%, 37%, 37%, 1.6%, and 0.1% as potent as (Thr59)IGF-I in inhibiting the binding of [125I]iodo-(Thr59)IGF-I to JEG-3 cells, respectively. Epidermal growth factor was ineffective. The half-maximal displacement of [125I]iodo-(Thr59)IGF-I by unlabeled (Thr59)IGF-I occurred at 11 +/- 2 ng/ml (mean +/- SEM) in both JEG-3 and BeWo cells. Scatchard analysis of the competitive binding data revealed linear plots indicating a single species of binding sites with an association constant of 0.8 X 10(9) M-1 for the binding of [125I]iodo-(Thr59)IGF-I to both cell lines. The binding capacity was 30,000 and 20,000 sites/cell for JEG-3 and BeWo cells, respectively. Chemical cross-linking of [125I]iodo-(Thr59)IGF-I to JEG-3 cells revealed two receptor complexes of 130K and 260K. Their formation was completely inhibited by an excess of unlabeled (Thr59)IGF-I or hIGF-II. Increasing amounts of insulin affected both labeled bands equally, suggesting that the 130K and 260K bands represent the monomer and dimer forms, respectively, of the ligand-binding alpha-subunit of type I IGF receptor. (Thr59)IGF-I, in a dose-dependent manner, stimulated uptake of nonmetabolizable alpha-[3H]aminoisobutyric acid by JEG-3 cells, showing that the receptor is biologically active. Our results demonstrate that choriocarcinoma cells possess functional high affinity type I IGF receptors and suggest that IGF-I is involved in the growth-regulating processes of JEG-3 and BeWo cells. These cells may provide a useful model to study the role of IGFs in trophoblast physiology.

Aminoisobutyric Acids↗

PubMed1988

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells.

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound [125I]iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of [125I] iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of [125I] iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with [125I]iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-[3H]aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells. We conclude that decidual 34 K IGF-BP inhibits the cellular binding and biological action of IGFs in JEG-3 cells. Our data show that JEG-3 cells represent a cell type that can produce IGF, but not IGF-BPs. These cells may thus provide a useful model system for a better understanding of autocrine growth regulation mediated by the IGFs.

Binding, Competitive↗

PubMed1988

Epidermal growth factor but not insulin-like growth factor-I potentiates adenosine 3',5'-monophosphate-mediated chorionic gonadotropin secretion by cultured human choriocarcinoma cells.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) regulate hormone production in several endocrine cells cultures. We have previously found that 12-O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C activator, potentiates the cAMP-mediated secretion of human CB (hCG) in cultured human choriocarcinoma cells. We have now studied whether EGF and IGF-I modify cAMP-mediated hCG secretion in JEG-3 cells, which possess high affinity receptors to these growth factors. EGF, TPA, and cholera toxin (CT), an activator of adenylate cyclase, stimulated the secretion of hCG in a concentration-dependent manner during a 24-h culture period. The maximal effective concentrations of EGF (10 ng/ml), TPA (10 ng/ml), and CT (1.0 ng/ml) exerted 2.3-, 2.4-, and 3.9-fold increase over unstimulated level, respectively. EGF and TPA potentiated the effect of CT on hCG secretion from 3.9- to 7.8-fold and from 3.9- to 14.8-fold, respectively. By contrast, IGF-I was ineffective. During a 24-h culture, EGF and TPA potentiated the effect of CT on cAMP accumulation 1.4-fold and 1.3-fold over the production of CT-treated cells. Time-course studies indicated that these effects on cAMP and hCG were detectable at 3 h and 6 h, and they continued to increase up to 48 h and 72 h, respectively. When added alone, EGF and TPA increased cAMP production y 2.0-fold and 2.5-fold over controls at 24 h. Again, IGF-I was ineffective. Moreover, EGF and TPA potentiated the effect of 8-bromo-cAMP (on hCG production to a similar extent than they did to CT-stimulated hCG production. The binding of [125I]iodo-EGF to the cells was not altered by a 48-h CT-treatment whereas the binding of [125I]iodo-IGF-I was increased by 2.1-fold above untreated cells. Our data show that both EGF and TPA potentiated the effect of CT on hCG secretion in JEG-3 cells, whereas IGF-I had no effect. Although EGF and TPA facilitated CT-stimulated cAMP accumulation, their site of action on cAMP-mediated hCG production is distinct from the adenylate cyclase or EGF-receptor level since EGF and TPA potentiated the hCG secretion stimulated by 8-bromo-cAMP and an increase in cAMP production did not alter the binding properties of EGF-receptor.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1987

Phorbol ester stimulates human granulosa-luteal cell cyclic adenosine 3', 5'-monophosphate and progesterone production.

Human granulosa-luteal cell production of cyclic adenosine 3', 5'-monophosphate (cAMP) and progesterone (P) were studied in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA specifically increased cAMP synthesis in a dose-dependent manner. A 7-fold increase occurred at a TPA concentration of 1 ng/ml. Time-course studies indicated that the increase in accumulation of cAMP into culture media became detectable at 4 h and continued up to 72 h. TPA also enhanced P synthesis, but the increase was statistically significant only at 72 h. Indomethacin prevented TPA-stimulated cAMP and P production. The results suggest that TPA stimulates granulosa-luteal cell cAMP and P production, and that the action of TPA is mediated by the increase in prostaglandin synthesis.

Corpus Luteum↗

PubMed1987

12-O-tetradecanoyl phorbol-13-acetate potentiates adenosine 3',5'-monophosphate-mediated chorionic gonadotropin secretion by cultured human choriocarcinoma cells.

The activation of protein kinase C and adenylate cyclase in the regulation of human CG (hCG) synthesis by cultured BeWo choriocarcinoma cells was studied. Both cholera toxin (CT), which activates adenylate cyclase, and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C activator, stimulated the secretion of hCG in a dose-dependent manner. During a 72-h culture, stimulation with the maximal effective concentration of CT (1.0 ng/ml) exerted a more pronounced increase (16-fold) in hCG synthesis than TPA (10 ng/ml) (2.8-fold), whereas the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate (100 ng/ml) was ineffective. When added together, TPA potentiated the effect of CT on hCG secretion (from 16- to 27-fold) and cAMP accumulation (from 36- to 54-fold) in the medium. TPA (1.0 ng/ml) also caused a 2.0-fold increase in basal cAMP production after 72 h. Time-course studies indicated that the effect of TPA on CT-induced cAMP and hCG productions became significant at 45 min and 6 h, respectively, from the beginning of stimulation. Proliferation of cells was not responsible for the responses, since the treatments only slightly increased the total protein content and did not alter the rate of incorporation of C3H3-methylated thymidine of the cells. Our results demonstrate that TPA potentiates CT-induced cAMP and hCG production in cultured human choriocarcinoma cells.

Adenylyl Cyclases↗