Human Choriocarcinoma JEG-3 Cells Research Articles

Structure of the VPATPD Gene Encoding Subunit D of the Human Vacuolar Proton ATPase

The HSD11B2 and VPATPD genes encoding the human kidney isozyme of 11β-hydroxysteroid dehydrogenase (11-HSD2) and subunit D of the vacuolar proton ATPase, respectively, are located on chromosome 16q22. They are transcribed from complementary DNA strands and their 3′ ends are only 0.5 kilobase apart. Because polymorphisms in HSD11B2 have been associated with hypertension and salt sensitivity, we characterized the human VPATPD gene. It spans 19 kb and consists of 8 exons. The encoded protein is 99.5% identical to mouse subunit D at the amino acid level. An alternating purine-pyrimidine tract is located in the 3′-untranslated region of VPATPD. On genotyping 17 hypertensive subjects, no length polymorphism was found. Although VPATPD and HSD11B2 are both expressed in kidney and placenta, they are regulated differently; forskolin upregulates HSD11B2 but not VPATPD in human choriocarcinoma JEG3 cells. The functional significance of the proximity of these two genes remains to be established.

Inhibition of malignant trophoblastic cell proliferation in vitro and in vivo by melatonin

Melatonin inhibited thymidine incorporation into human choriocarcinoma JEG-3 cells at physiological and pharmacological concentrations in the present study. Gene expression of MT 2 receptor, but not that of mt 1 receptor, was detected in JEG-3 cells by reverse transcription-polymerase chain reaction (RT-PCR). The gene expression profile of the two human melatonin receptor subtypes in JEG-3 cells was identical to that previously reported for JAr cells, whose proliferation had also been shown to be similarly inhibited by physiological and pharmacological concentrations of melatonin. In contrast, melatonin had no effect on thymidine incorporation into 3A-Sub-E cells (a transformed trophoblast cell line), in which gene expression of both receptor subtypes could not be detected. The data suggest that in human placental trophoblasts, a correlation may exist between MT 2 receptor gene expression and the direct anti-proliferative action of melatonin. Although melatonin has been reported to induce G1/S delay in cell cycle progression of JAr cells, no significant changes in the percentages of JEG-3 cells in different cell cycle phases upon melatonin treatment was recorded by flow cytometric analysis. This indicates that G1/S transition delay is probably not an important cellular mechanism in the direct anti-proliferative action of melatonin on human JEG-3 cells in vitro. Furthermore, melatonin inhibited the growth of both JAr and JEG-3 xenograft tumors in athymic nude mice, and prolonged the survival of those animals that developed choriocarcinoma. While the number of apoptotic tumor cells was not increased by melatonin, the pineal hormone induced significant decreases in the numbers of JAr and JEG-3 cells expressing proliferating cell nuclear antigen (PCNA) and cyclin A in the tumors. Taking into account both the in vitro and in vivo findings, it is likely that the inhibitory effect of melatonin on choriocarcinoma JAr and JEG-3 cell proliferation in vivo is largely a direct action of the hormone on the tumor cells.

A protein kinase C-independent pathway leading to c-Jun-dependent expression of 100-kDa Ras GTPase-activating protein in JEG-3 human choriocarcinoma cells.

Although the 100-kDa Ras GTPase-activating protein (p100 RasGAP) has been reported to exist specifically in human placental trophoblasts, the molecular mechanisms responsible for regulating its expression remain unclear. In this study we used okadaic acid, an inhibitor of serine/threonine phosphatase 1 and 2 A, as a probe to explore the signaling pathway regulating the expression of p100 RasGAP in JEG-3 human placental choriocarcinoma cells. Treatment of JEG-3 cells with okadaic acid provoked dose- and time-dependent stimulation of p100 RasGAP expression without marked modification of expression of p120 RasGAP, another isoform of RasGAP. Co-treatment of cells with okadaic acid and the protein kinase C activator, phorbol 12-myristate 13-acetate, exerted an additive effect on p100 RasGAP induction. Moreover, the response of the p100 RasGAP de novo synthesis to okadaic acid was not affected by the selective inhibitor of protein kinase C, GF 109203X. Thus this study identified a novel signaling pathway regulating p100 RasGAP expression, which is independent of protein kinase C. In addition, okadaic acid treatment resulted in the activation of ERK2 (p42 MAP kinase) and the induction of both c-Jun and c-Fos proteins without activating JNK (c-Jun NH2-terminal kinase). Significantly, blockade of c-Jun expression with antisense c-jun oligonucleotides suppressed p100 RasGAP expression. Taken together, it is concluded that okadaic acid induces the expression of p100 RasGAP protein in JEG-3 cells preceded by activation of ERK and AP-1 cascade, and that this okadaic acid-induced p100 RasGAP expression is independent of protein kinase C-mediated pathway but requires c-Jun/AP-1 function.

Activation of the cyclin D1 Gene by the E1A-associated Protein p300 through AP-1 Inhibits Cellular Apoptosis

The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at -954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1(-/-)) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1(+/+) fibroblasts but increased apoptosis in cyclin D1(-/-) cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G(1) analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties.

Evidence for a Novel RasGAP-Associated Protein of 105 kDa in both Mature Trophoblasts and Differentiating Choriocarcinoma Cells

A novel tyrosine-phosphorylated, RasGAP-associated protein of 105 kDa (p105) is found in normal human term placental trophoblasts, as well as in JEG-3 human choriocarcinoma cells induced to differentiate by okadaic acid (OA). This p105 RasGAP-associated protein is distinct from other RasGAP-associated proteins described so far, none of which has either a molecular size close to p105 or a trophoblastic cell origin. The p105 appears, accompanied by p120 and p100 RasGAP expression, after OA treatment of JEG-3 cells but is almost undetectable in the absence of stimulation. Moreover, the p105 is the first discovered RasGAP-associated protein bound to p100 RasGAP. The natural occurrence of the p105 in normal mature trophoblasts isolated from human term placenta suggests that it may be linked to the differentiation state of human trophoblasts. Hence, this p105 RasGAP-associated protein might be considered a marker of human trophoblast differentiation.

Prostaglandins and leukotriene B4 are potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 2 activity in human choriocarcinoma JEG-3 cells.

The 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is responsible for the inactivation of glucocorticoids. This is the predominant isozyme in the human placenta, where it is proposed to protect the fetus from high levels of maternal cortisol. In the present study, we examined the effects of eicosanoids on the activity of 11beta-HSD2 in human choriocarcinoma JEG-3 cells, a well-established model for placental trophoblasts. Treatment of JEG-3 cells for 24 h with either prostaglandin (PG) E2 or F2alpha attenuated 11beta-HSD2 activity ( approximately 40%). Paradoxically, indomethacin, an inhibitor of cyclooxygenases, inhibited (approximately 40%) rather than stimulated the activity of this enzyme. This indicated that the arachidonic acid metabolism may be diverted to other pathway(s), the products of which may inhibit 11beta-HSD2 activity. To determine whether the lipoxygenase pathways were involved, the cells were treated with nordihydroguaretic acid (NDGA), a blocker of all three (5-, 12-, and 15-) lipoxygenases. NDGA caused a 3-fold increase in 11beta-HSD2 activity. To further delineate which specific lipoxygenase pathway was involved, the cells were incubated with zileuton, a selective inhibitor of 5-lipoxygenase. This resulted in a similar increase in 11beta-HSD2 activity, suggesting that the products of this pathway (e.g., leukotrienes) may be involved. Given that leukotriene B4 (LTB4) is the most biologically active product of the 5-lipoxygenase pathway, we treated the cells with LTB4, which inhibited 11beta-HSD2 activity in a time- and dose-dependent manner with a maximal effect (60% reduction) at 10 nM for 9 h. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that 11beta-HSD2 mRNA levels were not altered by the addition of LTB4, PGE2, or PGF2alpha, indicating an effect at the posttranscriptional level. In conclusion, these results demonstrate that prostaglandins and LTB4 are potent inhibitors of 11beta-HSD2 activity in JEG-3 cells, suggesting that placental 11beta-HSD2 activity is modulated by these locally produced eicosanoids. This is the first time that the products of arachidonic acid metabolism have been found to regulate the activity of 11beta-HSD2.

Medroxyprogesterone acetate and dexamethasone are competitive inhibitors of different human steroidogenic enzymes.

Medroxyprogesterone acetate (MPA), a widely used progestin, can suppress the hypothalamic-pituitary-gonadal axis but can also directly inhibit gonadal steroidogenesis; the success of MPA as a treatment for gonadotropin-independent sexual precocity derives from its direct action on steroidogenic tissues. Dexamethasone, a widely used glucocorticoid, can suppress the hypothalamic-pituitary-adrenal axis, but its potential effect directly on the adrenal is unclear. Previous reports suggested that these two drugs may act on the initial steps in the rodent steroidogenic pathway; therefore, we investigated their abilities to inhibit the first three human enzymes in steroidogenesis: the cholesterol side-chain cleavage enzyme (P450scc), the 17alpha-hydroxylase/17,20-lyase (P450c17), and type II 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSDII). We found no effect of either drug on P450scc in intact human choriocarcinoma JEG-3 cells. Using microsomes from yeast expressing human P450c17 or microsomes from human adrenals, we found that dexamethasone inhibited P450c17 with a Ki of 87 micromol/L, which is about 1000 times higher than typical therapeutic concentrations, but that MPA has no detectable action on P450c17. Using microsomes from yeast expressing human 3betaHSDII, we found that this enzyme has indistinguishable apparent Km values of 5.2-5.5 micromol/L and similar maximum velocities of 0.34-0.56 pmol steroid/min x microg microsomal protein for the three principal endogenous substrates, pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone. In this system, MPA inhibited 3betaHSDII with a Ki of 3.0 micromol/L, which is near concentrations achieved by high therapeutic doses of 5-20 mg MPA/kg x day. These data establish the mechanism of action of MPA as an inhibitor of human steroidogenesis, and are in contrast with the results of earlier studies indicating that MPA inhibited both P450c17 and 3betaHSD in rat Leydig cells. These studies establish the "humanized yeast" system as a model for studying the actions of drugs on human steroidogenic enzymes and suggest that 3betaHSDII may be an appropriate target for pharmacological interventions in human disorders characterized by androgen excess or sex steroid dependency.

Retinoic acid stimulates the expression of 11beta-hydroxysteroid dehydrogenase type 2 in human choriocarcinoma JEG-3 cells.

The syncytiotrophoblasts of the human placenta express high levels of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the enzyme responsible for the inactivation of glucocorticoids. It has been proposed that the placental 11beta-HSD2 serves as a barrier to protect the fetus from high levels of maternal cortisol. To examine the hypothesis that nutritional signals regulate the expression of 11beta-HSD2 in placental syncytiotrophoblasts, we investigated the effects of retinoic acids (RAs), the major metabolites of vitamin A, on the expression of 11beta-HSD2 using human choriocarcinoma JEG-3 cells as a model. This trophoblast-like cell line displays a number of functional similarities to the syncytiotrophoblast. Treatment for 24 h with all-trans RA (1-1000 nM) resulted in a dose-dependent increase in 11beta-HSD2 activity with a maximal effect (increase to 3-fold) at 100 nM. The effect of all-trans RA (100 nM) was also time-dependent in that the effect was detectable at 6 h and reached its maximum by 48 h. Similar increases in 11beta-HSD2 activity were observed when the cells were treated with 9-cis RA. Results from semi-quantitative reverse transcription-polymerase chain reaction demonstrated that there was a corresponding increase in 11beta-HSD2 mRNA after RA treatment. Moreover, treatment with actinomycin D (100 ng/ml) abrogated the increase in 11beta-HSD2 mRNA induced by RA, indicating an effect on transcription. In conclusion, the present study has demonstrated for the first time that RA, at physiological concentrations, induces 11beta-HSD2 gene expression and enzyme activity in JEG-3 cells. If this occurs in vivo, the present finding suggests that high expression of 11beta-HSD2 in the human placenta may be maintained, at least in part, by dietary intake of vitamin A.

Establishment of bovine blastocyst-derived trophectoderm and endoderm cell lines: Use of interferon tau and serum protein expression as respective in vitro markers

Interferon tau (IFNT) is the pregnancy recognition protein in all ruminants, and its expression is restricted to trophoblast cells. Interferon tau production increases as the conceptus elongates; however, its expression is downregulated soon after the initiation of conceptus attachment to the uterine epithelium. Our previous study identified that among 8 bovine IFNT genes, only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the periattachment period. To characterize whether Hippo signaling including a transcription cofactor yes-associated protein (YAP) was involved in the IFNT regulation, we examined the expression and effects of YAP and/or TEAD in human choriocarcinoma JEG3 and bovine trophoblast CT-1 cells, and in bovine conceptuses obtained from day 17, 20 or 22 pregnant animals (pregnant day 19.5 = day of conceptus attachment to the endometrium). YAP was expressed in bovine conceptuses and transfection of YAP or TEAD4, a transcription factor partner of YAP, expression plasmid increased the luciferase activity of IFNT2 and IFN-tau-c1 reporter plasmids in JEG3 cells. In the presence of YAP expression plasmid, TEAD2 or TEAD4 expression plasmid further upregulated transcriptional activity of IFNT2 or IFN-tau-c1 constructs, which were substantially reduced in the absence of the TEAD-binding site on IFNT2 or IFN-tau-c1 promoter region in JEG3 cells. In CT-1 cells, treatment with TEAD2, TEAD4, or YAP small-interfering RNA downregulated endogenous IFNT expression. It should be noted that TEAD2 and TEAD4 were predominantly localized in the nuclei of trophectoderm of Day 17 conceptuses, but nuclear localization appeared to be lower in those cells of conceptuses on days 20 and 22 of pregnancy. Moreover, the binding of TEAD4 to the TEAD-binding site of the IFN-tau-c1 promoter region in day 17 conceptuses was less in day 20 and 22 conceptuses. Furthermore, the level of YAP phosphorylation increased in day 20 and 22 conceptuses. These results indicated that although YAP/TEAD had the ability to up-regulate IFNT gene transcription on day 17, IFNT2 or IFN-tau-c1 was down-regulated following changes in the localization of TEAD2 and TEAD4 from the nucleus to the cytoplasm and increases in phosphorylation and degradation of YAP. These data suggest that TEAD relocation and/or YAP degradation following its phosphorylation down-regulates IFNT gene transcription after conceptus attachment to the uterine endometrium.

Jeg-3 human choriocarcinoma-induced immunosuppression: downregulation of interleukin-2, interleukin-2 receptor alpha-chain, and its Jak/Stat signaling pathway.

The mechanisms of the immunosuppressive and immunosuppression-inducing capacities of Jeg-3 human choriocarcinoma cell line supernatants (HCSs) are not yet completely understood. The influence on interleukin (IL)-2, IL-4 and interferon (IFN)-gamma production; IL-2 receptor (IL-2R) alpha-, beta-, and gamma-chain; and the signaling pathway molecules Janus kinase (Jak)1, Jak3, signal transducers and activators of transcription (Stat)1, Stat3, and Stat5 should be investigated. For assessment of IL production, whole peripheral venous blood from healthy donors was stimulated with phorbol-myristate-acetate and ionomycine. Secretion of ILs was blocked with monensine. Intracellular ILs were analyzed by flow cytometry. For IL-2R and signaling pathway molecule analysis, peripheral blood lymphocytes were stimulated with phytohemagglutinin (PHA). IL-2R chains were measured by flow cytometry, and Jaks/Stats by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Phorbol-myristate-acetate and ionomycine strongly increase the percent-age of IL-2+ cells; an additional 50% HCSs significantly suppresses the percentage to, or below the level of unstimulated cells. IFN-gamma production is strongly decreased by HCSs in some cases, but not in others. PHA stimulates IL-2R alpha-, beta-, and gamma-chain expression and their signaling pathway molecules Jak1, Jak3, Stat1, Stat3, and Stat5. 50% HCS downregulates the alpha-chain and slightly upregulates the beta-chain. Jak1, Jak3, Stat1, Stat3, and Stat5 expression is suppressed approximately to, or below the level of unstimulated cells. HCS forcefully blocks the production of IL-2; the IL-2R alpha-chain; and Jak1, Jak3, Stat1, Stat3, and Stat5 expression. The observed phenomena might be caused by downregulation of an IL-2R regulation gene, and might play a key role in the expansion of choriocarcinoma, and possibly in the survival of the fetal allograft.

Mitogen-activated protein kinase is involved in epidermal-growth-factor-regulated protein phosphorylation in nuclear membranes isolated from JEG-3 human choriocarcinoma cells.

The treatment of highly purified nuclear membranes isolated from JEG-3 human choriocarcinoma cells with epidermal growth factor (EGF) resulted in an increase of the receptor autophosphorylation and the phosphorylation of several other proteins, including 44-kDa, 34-kDa and 24-kDa proteins, and a decrease in the phosphorylation of a 40-kDa protein. The kinetics of phosphorylation and the use of RG-13022, a selective inhibitor of EGF-receptor kinase activity, suggested that receptor activation was necessary for the phosphorylation response of the other proteins. Tyr was exclusively phosphorylated in the EGF receptor and 24-kDa proteins, Tyr and Thr were phosphorylated in the 44-kDa protein, and Ser was phosphorylated in the 34-kDa protein and dephosphorylated in the 40-kDa protein. The molecular size, Thr/Tyr phosphorylation, immunoprecipitation and enzymatic activity towards myelin basic protein suggested that the 44-kDa protein was a mitogen-activated protein (MAP) kinase. The EGF treatment not only increased phosphorylation but also catalytic activity of MAP kinase and these increases were prevented by the addition of RG-13022. In summary, this report demonstrates that target cell nuclei contain EGF receptors, which use the MAP kinase signaling pathway.

Effects of Some Persistent Halogenated Environmental Contaminants on Aromatase (CYP19) Activity in the Human Choriocarcinoma Cell Line JEG-3

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a technical PCB mixture (Aroclor 1016), and a technical toxaphene mixture (Camphechlor) on aromatase (CYP19) activity were investigated in human choriocarcinoma JEG-3 cells. After 18 h incubation with TCDD, PCB126, Aroclor 1016 or toxaphene, ethoxyresorufin-O-deethylase (EROD), and aromatase activity were determined. To exclude serum effects, incubations were carried out with or without fetal calf serum in the medium. EROD activity was induced by both TCDD and PCB126 in the presence or absence of serum, which indicates that JEG-3 cells are responsive toward dioxin-like chemicals. Neither Aroclor 1016 nor toxaphene affected EROD activity in these cells. Calculated EC50 values for induction of EROD activity were 0.71 and 0.40 nmfor TCDD, and 48 and 20 nmfor PCB126 in presence or absence of serum, respectively. Incubation with TCDD or PCB126 with or without serum caused a concentration-dependent decrease in the aromatase activity of up to 4.9-fold. Calculated EC50 values for this effect were 52 pmand 13 nmfor TCDD, and 75 and 48 nmfor PCB126 in the presence and absence of serum, respectively. Aroclor 1016 and toxaphene had no effect on aromatase activity at concentrations up to 1.0 μmfor Aroclor 1016 or 3.0 μmfor toxaphene. These results show that aromatase activity can be decreased in a concentration dependent way within the same range where EROD activity is increased. In view of these results, possible effects of dioxin-like compounds on estrogen producing and androgen target cells should be studied in more detail.

Regulation of cytochrome P450 cholesterol side-chain cleavage, 3 β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type 1 and estradiol-17 β-hydroxysteroid dehydrogenase mRNA levels by calcium in human choriocarcinoma JEG-3 cells

In human placenta the cytochrome P450 side-chain cleavage (P450scc) and 3 β-hydroxysteroid dehydrogenase type 1 (3 β-HSD-1) convert cholesterol and pregnenolone producing progesterone, whereas 17 β-hydroxysteroid dehydrogenase type 1 (17 β-HSD-1) mediates the interconversion of estrone and estradiol. We have examined the effects of calcium on phorbol ester- and cAMP-induced P450scc, 3 β-HSD-1 and 17 β-HSD-1 mRNAs in human JEG-3 cells. A23187 increased in a dose-dependent fashion in the 1.3 kb 17 β-HSD-1 mRNA whereas a weaker increase followed by a gradual depletion effect of A23187 was observed on 3 β-HSD-1 mRNA. No significant effect of A23187 on P450scc mRNA was observed. Using 0.50 μM of A23187 the induction of 3 β-HSD-1 and 17 β-HSD-1 mRNAs was maximum within about 6 h whereas P450scc mRNA levels stayed unaffected throughout the time-course period. The action of A23187 was synergistic on cAMP-stimulated 17 β-HSD-1 mRNA levels, while in a dose-dependent manner A23187 progressively depleted 3 β-HSD-1 and P450scc mRNA abundance probably by activation of a calcium-/calmodulin-dependent phosphodiesterase. On the phorbol 12-myristate, 13-acetate (PMA)-stimulated 3 β-HSD-1, 17 β-HSD-1 and P450scc mRNA levels only the lowest concentration of A23187 potentialized the PMA effect on the 17 β-HSD-1 mRNA levels. Using thapsigargin (TG), a cell-permeable sesquiterpene lactone that releases calcium by inhibiting sarco/endoplasmic reticular calcium-ATPase, our data indicated the presence in JEG-3 cells of TG-sensitive and TG-insensitive calcium-ATPases regulating 3 β-HSD-1 and 17 β-HSD-1 mRNA levels. These results emphasized the complexity of calcium contribution with the protein kinase A and C pathways in the regulation of P450scc, 3 β-HSD-1 and 17 β-HSD-1 mRNA levels. In addition, the different sensitivity of these genes to calcium suggest they could be activated by different subclasses of PKCs.

Thyroid hormone activates transcription from the promoter regions of some human nuclear-encoded genes of the oxidative phosphorylation system

Thyroid hormone (T3) modulates the mRNA levels for cytochrome c1 and the adenine nucleotide translocator-2 (ANT2) in adult rat liver. Here we show that T3 activates expression of a reporter gene driven from the human cytochrome c1 and ANT2 promoters transfected into human choriocarcinoma JEG3 cells. By contrast, the human F1-ATPase β-subunit promoter responded marginally, thus providing a pattern of differential expression similar to that earlier observed in rats in vivo. T3-activation is dependent on co-expression of the thyroid hormone receptor (TR α1). Co-expression of both the TR and RXR receptors had no additional effect. Transient transfection of deletion constructs showed that T3 activation is retained by the proximal regions of the cytochrome c1 and ANT2 promoters, and, in the case of cytochrome c1, is lost upon removal of a fragment containing the transcription initiator ((nucleotides) (nt) +1 to +100). The promoter regions supporting T3-activation of the reporter genes appear to lack strong DNA binding sites for TR and retinoid X receptor (RXR).

Regulation of aromatase expression in the ovary and placenta: a comparison between two species

The conversion of C19 steroids to estrogens is catalysed by aromatase P450 (P450arom; product of the CYP19 gene). Tissue sites of expression include the gonads and brain; however, in a small subset of mammals, P450arom is also expressed in the placenta. In humans, gonadal expression employs a promoter proximal to the start site of translation, whereas expression in the placenta relies on a promoter which is distal to this site. We characterized the bovine CYP19 gene in both the ovary (OV) and placenta (PL), to determine if this method of regulation of tissue-specific expression is employed in other species. Both bovine and human species express P450arom in these tissues, however, the pattern of expression is very different. In both humans and cattle, P450arom is expressed in the granulosa cells of the ovary, however, after ovulation only the luteinized granulosa cells of the human continue to express P450arom. There is a high degree of sequence identity (>70%) shared between humans and cattle in the OV-specific 5′-flanking DNA, whereas little identity (<40%) was found between humans and cattle in the PL-specific DNA. Promoters and 5′-flanking regions of human and bovine CYP19 genes were subcloned into a luciferase (LUC) vector. Bovine PL-specific constructs transfected into JEG-3 human choriocarcinoma cells failed to express LUC activity. When OV-specific constructs were transfected into luteinized bovine granulosa cells, there was no change in LUC activity in cells transfected with the bovine constructs after treatment with forskolin, whereas all of the corresponding human constructs expressed LUC activity. The bovine 5′-flanking DNA lacks a cAMP-responsive element-like sequence (CLS) critical for cAMP-stimulated transcription of P450arom in the human ovary. With the absence of this CLS sequence in the bovine gene, there appears to be little enhancement of transcription by cAMP in the portion of the 5′-flanking region studied so far. When the analogous region of the bovine promoter was mutated to the human CLS, only a partial restoration of LUC activity was observed. An additional element therefore appears to be important in preventing the full expression of the bovine CYP19 gene in luteinized granulosa cells.

Transcription of the human genes for cytochrome P450scc and P450c17 is regulated differently in human adrenal NCI-H295 cells than in mouse adrenal Y1 cells.

Human NCI-H295 cells, which express all of the genes for the steroidogenic enzymes in a hormonally regulated fashion, should be an ideal system in which to study the transcriptional regulation of these genes. Using deletional promoter/reporter constructions for the human P450scc and P450c17 genes, we identified the regions conferring basal and cAMP-induced transcription of these two genes in NCI-H295 human adrenal cells. In the P450scc gene, both basal and cAMP-induced transcriptional activation elements lie within the first 79 bp upstream (-79) from the transcriptional start site. In the P450c17 promoter, both basal and cAMP-responsive elements lie within the first upstream 63 bp, and a second basal element lies between -184 and -206 bp. The locations of these elements are substantially different from the locations of elements that appear to be functionally equivalent when these human gene promoters are transfected into mouse adrenal Y1, mouse testicular MA-10, or human choriocarcinoma JEG-3 cells. These data indicate that the transcriptional regulation of these genes in their native species and cell type differs substantially from their regulation in cells from other species and tissues, and suggests that the results from transfection experiments examining genes for steroidogenic enzymes in heterologous cells may not reflect events in vivo.

Benzo(a)pyrene, but Not 2,3,7,8-Tetrachlorodibenzo-p-dioxin, Alters Cell Proliferation and c-Myc and Growth Factor Expression in Human Placental Choriocarcinoma JEG-3 Cells

This study compared the effects of benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two aryl hydrocarbon receptor (AhR) agonists, on proliferation and gene expression in the human placental choriocarcinoma JEG-3 cell line. BaP significantly inhibited [3H]thymidine incorporation, whereas no effect of TCDD was observed over a 7 day period. TCDD and BaP both showed induction of cytochrome P450 1A1 (CYP1A1), whereas only BaP caused a significant loss of EGFRs. Exposure to 10 μM BaP significantly increased the steady state mRNA level of transforming growth factor (TGF)-β1, while the c-myc mRNA levels were decreased by 61%. In contrast, TCDD showed no changes in mRNA levels for TGF-β1 and c-myc. Thus, although both compounds induce CYP1A1, only BaP inhibits cell proliferation which is correlated with disruption of expression of significant regulators of trophoblast growth.

Analysis of the promoter of the NAD+ dependent 11β-hydroxysteroid dehydrogenase (HSD11K) gene in JEG-3 human choriocarcinoma cells

The NAD+ dependent (K or type 2) isozyme of 11β-hydroxysteroid dehydrogenase oxidizes glucocorticoids and thus prevents them from occupying mineralocorticoid receptors. Mutations in the HSD11K (HSD11B2) gene encoding this isozyme cause a genetic form of hypertension, the syndrome of apparent mineralocorticoid excess (AME). This isozyme is expressed at high levels in placenta and kidney but is undetectable in liver. We have now analyzed the proximal 1788 nucleotides (nt) of the 5′ flanking region of the HSD11K gene to identify transcriptional regulatory elements that are active in JEG-3 human choriocarcinoma cells. Using luciferase reporter constructs, the region from −2 to −330 nt relative to the initial ATG codon was identified as an essential region for basal transcription of the HSD11K gene. Two segments in this region, −278 to −257 and −215 to −194, were protected in DNase 1 footprinting analysis. Both segments have consensus binding sites for the Spl transcription factor. Gel shift assays of these segments show several DNA-protein complexes using JEG-3 nuclear extract. Only the slowest migrating complex was competed by an antiserum to Spl. These results suggest that the two Spl sites, either alone or in combination, are essential for transcription of the HSD11K gene in JEG-3 cells.

Nuclease sensitivity of the human growth hormone-chorionic somatomammotropin locus in pituitary and placenta suggest different mechanisms for tissue-specific regulation

The five human growth hormone (GH) and chorionic somatomammotropin (CS) genes are located at a single locus on chromosome 17. These genes share extensive nucleotide sequence similarity (∼94%) even in their flanking DNA. yet GH-N is expressed efficiently in the pituitary under the control of the pituitary-specific factor GHF-1 Pit-1 and the remaining CS-A, CS-B, CS-L and GH-V genes are transcriptionally active in the placenta. Despite this specificity in vivo, a truncated CS-A promoter can bind GHF-1 Pit-1 and allow CS-A promoter activity in pituitary cells in vitro. With a view to assessing whether the placental genes of the GH CS locus possess a different chromatin structure in the pituitary and are, thus, less transcriptionally active than the GH-N gene, we have compared the DNAase I sensitivity of GH CS in isolated pituitary and placenta cell nuclei. Our data indicate that these genes are equally sensitive in isolated human pituitary nuclei. By contrast, the CS-A, CS-B and CS-L genes were significantly ( P < 0.05) more sensitive than the GH-N gene in isolated human placenta nuclei. Although just not significant, the GH-V gene was slightly more sensitive than the GH-N gene. This pattern was also seen with nuclei from human choriocarcinoma BeWo and JEG-3 cells, which express low and extremely low levels of CS RNA, respectively, but was distinct from the pattern observed in the non placental human cervical carcinoma HeLa cell line. These data indicate that the inactivity of the CS genes in the pituitary does not correlate with a ‘closed’ chromatin structure. However, they are consistent with a role for a more ‘open’ chromatin conformation in placenta-specific expression, but not necessarily high levels of transcriptional activity.

Okadaic acid, a protein phosphatase inhibitor, mimics PMA effect on induction of differentiation in JEG-3 human placental choriocarcinoma cells

Okadaic acid, a protein phosphatase inhibitor, mimics PMA effect on induction of differentiation in JEG-3 human placental choriocarcinoma cells