Human Placental Choriocarcinoma Cell Line Research Articles

Transcriptional regulation of human placental corticotropin-releasing factor by prostaglandins and estradiol.

The mechanism of labor initiation in humans has not been completely elucidated. Prostaglandins, estrogens, and corticotropin-releasing factor (CRF) have all been shown to affect uterine myocytes and enhance uterine contractility. There are also indications that these uterine regulators have additional effects on other sites involved in labor and that they may act in concert or, perhaps, by regulating each other. Therefore, we evaluated the CRF promoter for transcriptional regulation by prostaglandins and estrogens. Human placental choriocarcinoma cell lines were transfected with CRF-luciferase reporter genes and treated with prostaglandins. Prostaglandin E2 (PGE2), but not prostaglandin F2alpha (PGF2alpha), stimulated CRF-luciferase expression in choriocarcinoma cell lines via a cAMP-dependent pathway. A combination of transfections and in vitro binding studies tested for potential regulation of CRF by estrogen receptor (ER). ER neither regulated the CRF promoter nor interacted with steroid response half-sites from the CRF promoter. Our results provide a molecular regulatory link between PGE2 and CRF, two compounds that enhance uterine contractile function. Combined with the stimulation of prostaglandin release by CRF, these data support a potentially important "feed-forward" regulatory loop involving CRF and PGE2 in parturition. In contrast, we found no evidence for direct effects of estrogens or PGF2alpha on CRF transcription.

Tyrosine phosphorylation-and epidermal growth factor-dependent regulation of the sodium-coupled amino acid transporter B 0 in the human placental choriocarcinoma cell line JAR

We have recently cloned an amino acid transporter from the human placental choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with characteristics known to be associated with the amino acid transport system B 0 (R. Kekuda, P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, S. Sinha, T.L. Yang-Feng, F.H. Leibach, and V. Ganapathy, J. Biol. Chem. 271, 18657–18661, 1996). The presence of the amino acid transport system B 0 (ATB 0) has however not been previously described in these cells by functional studies. In the present investigation, we have obtained evidence for the existence of ATB 0 in JAR cells and delineated the functional characteristics of the transporter. The identifying characteristics include Na +-dependence and preference for neutral amino acids. In addition, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB 0. Treatment of the cells with the neuroprotective agent aurintricarboxylic acid (ATA) for 16 h leads to a significant increase in ATB 0 activity. This increase is associated with enhanced maximal velocity of the transporter and with increased steady state levels of the transporter mRNA. The effect of ATA is blocked by the tyrosine kinase inhibitor genistein. ATA treatment results in increased tyrosine phosphorylation of two major proteins, 180 kDa and 140 kDa in size. The 180 kDa protein is likely to be the epidermal growth factor (EGF) receptor because exposure of the cells to EGF also leads to enhanced tyrosine phosphorylation of a protein of similar molecular size. Furthermore, the effects of ATA on ATB 0 activity and on ATB 0 mRNA levels can be reproduced by EGF. Treatment of the cells with EGF for 24 h results in a significant increase in ATB 0 activity and this effect is associated with an increase in the maximal velocity of the transporter and with an increase in the steady state levels of the transporter mRNA. These data suggest that ATA influences ATB 0 activity in JAR cells most likely by activating the EGF receptor through tyrosine phosphorylation. It is concluded that the human placental choriocarcinoma cells functionally express the amino acid transport system B 0 and that the expression of the system in these cells is stimulated by EGF.© 1997 Elsevier Science B.V. All rights reserved.

Cloning of the Sodium-dependent, Broad-scope, Neutral Amino Acid Transporter Bo from a Human Placental Choriocarcinoma Cell Line

We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na+-dependent amino acid transport system with preference for zwitterionic amino acids. Anionic amino acids, cationic amino acids, imino acids, and N-methylated amino acids are excluded by this system. These characteristics are identical to those described for the amino acid transporter Bo. When expressed in Xenopus laevis oocytes that do not have detectable endogenous activity of the amino acid transporter Bo, the cloned transporter increases alanine transport in the oocytes severalfold and induces alanine-evoked inward currents in the presence of Na+. The cDNA codes for a polypeptide containing 541 amino acids with 10 putative transmembrane domains. Amino acid sequence homology predicts this transporter (hATBo) to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located the ATBo gene to human chromosome 19q13.3.

Cloning of neutral amino acid transporter B 0 from a human placental choriocarcinoma cell line

Cloning of neutral amino acid transporter B 0 from a human placental choriocarcinoma cell line

Modulation of the activity of amino acid transport system L by phorbol esters and calmodulin antagonists in a human placental choriocarcinoma cell line

We investigated the regulation of the activity of amino acid transport system L in the JAR human placental chornocarcinoma cell line by agents which are known to modulate the activities of three different classes of protein kinases, A-kinase. C-kinase and CaM-kinase. The system L activity was measured by determining the uptake of leucine in these cells, grown as confluent monolayers. Leucine uptake in these cells was predominantly Na +-independent, was stimulated by lowering the extracellular pH and was inhibited by hydrophobic neutral amino acids. These characteristics demonstrate that uptake of leucine in this cell line occurs primarily via system L. Treatment of the cells with cholera toxin and forskolin, agents which are known to elevate intracellular cAMI levels, did not have any effect on the activity of system L. 4β-Phorbol 12-myristate 13-acetate, an activator of C-kinase, but not the inactive analogue 4α-phorbol 12,13-didecanoate, caused a significant stimulation of system L. The involvement of C-kinase in the phorbol ester-induced stimulation was supported by the finding that staurosporine, an inhibitor of C-kinase, effectively blocked the stimulation. Calmodulin antagonists, calmidazolium, W-7 and CGS 9343 B stimulated system L activity markedly. The potency of these antagonists was in the following order: calmidazolium > CGS 9343 B > W-7. This stimulatory effect was specific for system L because systems A and ASC were not stimulated by these agents. The stimulation caused by these agents was primarily due to an increase in the maximal velocity, the apparent K m of the system being only minimally affected. It is concluded that the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line is stimulated by C-kinase, inhibited by CaM-kinase and unaffected by A-kinase.

Selective impairment of taurine transport by cyclosporin A in a human placental cell line.

We investigated, using a human placental choriocarcinoma cell line as a model, the effects of the immunosuppressive drug cyclosporin A on several placental transport systems mediating the transfer of glucose and amino acids from mother to fetus. The results of the investigation show that the transport system responsible for the transfer of taurine is selectively impaired by the drug, whereas the other transport systems are either stimulated or not affected. The inhibitory effect of the drug on taurine transport appears to be due to interference with calmodulin-dependent processes because calmodulin antagonists such as W-7, calmidazolium, and CGS 9343B mimic the effects of cyclosporin A. FK506, another immunosuppressive drug that is currently undergoing clinical trials, does not have this inhibitory effect.

Relative sensitivity to inhibition by cimetidine and clonidine differentiates between the two types of Na(+)-H+ exchangers in cultured cells.

Available evidence indicates that there are two types of Na(+)-H+ exchangers, type A (housekeeping type) and type B (epithelial or apical type), in mammalian cells. We have recently reported, using isolated membrane vesicles, that these two types can be differentiated by their relative sensitivities to inhibition by clonidine and cimetidine [Kulanthaivel, Leibach, Mahesh, Cragoe & Ganapathy (1990) J. Biol. Chem. 264, 1249-1252]. The present study was undertaken to determine whether this approach is also effective in cultured cells. The JAR human placental choriocarcinoma cell line and the opossum kidney (OK) cell line, when grown as confluent monolayer cultures on an impermeable plastic support, express Na(+)-H+ exchanger activity which is measurable by determining Na+ uptake into the cells from the culture medium. The JAR cell Na(+)-H+ exchanger was found to be about 100 times more sensitive to inhibition by dimethylamiloride than the OK cell Na(+)-H+ exchanger. Inhibition studies with clonidine and cimetidine were able to differentiate between these two exchangers very clearly. Cimetidine was 18 times more potent than clonidine in inhibiting the JAR cell Na(+)-H+ exchanger. In contrast, clonidine was at least 8 times more potent than cimetidine in inhibiting the Na(+)-H+ exchanger of the OK cell. The results show that the JAR cell expresses the type A Na(+)-H+ exchanger, whereas the OK cell expresses the type B Na(+)-H+ exchanger. This approach also proved to be very effective in correctly identifying the type of Na(+)-H+ exchanger in a third cell line (HeLa). It is concluded that the relative susceptibility to inhibition by clonidine and cimetidine offers an easy and efficient means of differentiating between the two types of Na(+)-H+ exchangers in cultured cells.

Expression and cyclic AMP-dependent regulation of a high affinity serotonin transporter in the human placental choriocarcinoma cell line (JAR)

The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5-fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation.

Transport of taurine and its regulation by protein kinase C in the JAR human placental choriocarcinoma cell line

The JAR human placental choriocarcinoma cell line transports taurine, concentrating it over 1000-fold inside the cell. The transport system is energized by a Na+ gradient and exhibits an absolute requirement for Cl-. Neutral beta-amino acids such as beta-alanine and hypotaurine effectively compete with the system, whereas neutral alpha-amino acids such as alanine, leucine and alpha-aminoisobutyric acid do not. The transport system interacts with gamma-aminobutyric acid to an appreciable extent. Kinetic analysis reveals that the taurine transport system in this cell line is of a high-affinity and low-capacity type (apparent dissociation constant 2.3 +/- 0.3 microM; maximal velocity 88.5 +/- 5.0 pmol/3 min per mg of protein). Pretreatment of the JAR choriocarcinoma cells with phorbol 12-myristate 13-acetate results in the inhibition of the taurine transport system in a dose-dependent manner. The inhibition is blocked by co-treatment of the cells with staurosporine, an inhibitor of protein kinase C. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, has no effect on the transport system. These data show that the choriocarcinoma cells express a taurine transporter with characteristics similar to those of the taurine transporter described in the normal human placenta, and that the activity of the transporter in these cells is under the regulatory control of protein kinase C.

DNA Sequences Involved in the Transcriptional Activation of a Human Placental Lactogen Gene

To identify regulatory elements in the promoter of a human placental lactogen gene (hPL3) that are important for its transcriptional activation, sequences 5' to the start of transcription were linked to the reporter gene chloramphenicol acetyltransferase (CAT) and transiently transfected into JEG-3 cells, a human placental choriocarcinoma cell line. In the presence of the hPL3 enhancer, deletion of the promoter sequence between -142 and -129 basepairs resulted in an 8-fold decrease in CAT activity. Similar results were seen with the SV40 enhancer and the hPL3 promoter in HepG2 liver cells. Nuclear proteins from HepG2, HeLa, and JEG-3 cells formed specific binding complexes with this region of the hPL3 promoter by a gel mobility shift assay, indicating that the DNA-binding protein was not tissue specific. The -142 to -129 basepair region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. An oligonucleotide containing Sp1-binding sites specifically competes for proteins binding the hPL3 promoter, and the methylation interference pattern is similar to that for an Sp1-binding site. This suggests that the hPL3 promoter binds Sp1- or an Sp1-like trans-acting factor, and this binding site is important for transcriptional regulation by the hPL3 enhancer in PL-producing cells.