Expression in Human Trophoblast and Choriocarcinoma Cell Lines, BeWo, Jeg-3 and JAr of Genes Involved in the Hepatobiliary-like Excretory Function of the Placenta☆
Introduction
The placenta plays a crucial role transferring a large variety of substances, including oxygen, nutrients, vitamins, hormones, etc., from the mother to the fetus. On the other hand, owing to the immaturity of the fetal liver, the placenta also constitutes the main route of elimination of potentially toxic compounds produced by fetal metabolism, such as cholephilic compounds, so-called because they are efficiently eliminated in bile by the liver in the adult. These compounds include bile acids and biliary pigments, which are synthesized by the fetal liver from very early on in gestation [1], and hence they must be transferred toward the maternal blood across the placenta [2].
The vectorial characteristics of the overall mechanisms involved in the hepatobiliary-like excretory role of the placenta [3] also constitute a barrier that provides protection for the conceptus against any potentially toxic endogenous compounds or xenobiotics that may reach the maternal side of the placenta [4]. In addition to the transport machinery, the placental barrier also includes enzymes able to perform many different detoxification reactions [5].
The trophoblast constitutes the outermost layer of the chorionic villi and is a key element in the placental barrier. Understanding the role of the uptake and export transporters involved in the excretory function of the trophoblast and the mechanisms of regulation of these proteins is crucial for evaluating the level of protection afforded by the placenta to the fetus and for elucidating the potential pharmacological usefulness or toxicological danger of drugs and other xenobiotics.
In light of the above, the present study was undertaken following two lines of interest: (i) to gain insight into the molecular bases of the mechanisms involved in the hepatobiliary-like excretory function of the placenta and, (ii) to characterize three choriocarcinoma cell lines as potentially suitable “in vitro” models for use in further studies addressing the regulation of the expression and function of the plasma membrane transporters involved in this aspect of the physiology and pharmacology of the placenta.
In this respect, several commercially available immortalized trophoblast cell lines, including BeWo, Jeg-3 and JAr, have been previously used as models for studying the transport/barrier function of the placenta. These lines were established from human choriocarcinoma cells (hCC) derived from first trimester trophoblast. The similarities between hCC and trophoblast support the use of these cells as valid and suitable models to study different aspects of trophoblast physiology and pharmacology. This is of particular interest in view of the difficulty involved in obtaining large amounts of freshly isolated trophoblast cells [6].
In spite of similarities among these hCC in several aspects – they are able to synthesize chorionic gonadotropin and steroids – they differ markedly in other characteristics, such as their proliferative activity and degree of differentiation. Thus, BeWo and JAr cells are less differentiated than Jeg-3 cells, but they have higher rates of proliferation [6], [7], [8]. Therefore, in order to use them in further investigations it is important to characterize their expression pattern of the transporters and nuclear receptors involved in the hepatobiliary-like excretory role of the placenta, because this information will be useful to decide which cell line should be chosen in each case to carry out future studies on particular aspects of this placental function.
Section snippets
Materials
Porcine pancreas trypsin, ethylene diamino tetra-acetic acid (EDTA), Percoll, and density marker beads were purchased from Sigma–Aldrich Quimica S.A. (Madrid, Spain). Dispase II and DNAse I were purchased from Roche (Barcelona, Spain). [6,7-3H]-estradiol 17β-d-glucuronide ([3H]-E217βG, 40.5 μCi/nmol) was from NEN (Pacisa + Giralt, Madrid, Spain). All other chemicals were obtained from Merck (Barcelona, Spain) or Sigma–Aldrich.
Isolation of human trophoblast cells
Isolated trophoblast cells (hTr) were purified from 12 human term
Expression of BSEP in human trophoblast
To elucidate the expression of genes involved in hepatobiliary function in human trophoblast, the presence of mRNA for a panel of selected proteins was investigated in placental cells by conventional PCR, followed by sequencing (Table 1) of the cDNA recovered from an agarose gel after electrophoretic separation. Although the expression in human placenta or hCC of some of the proteins studied here has been already reported, we have included them in the present study to have a complete panel for
Discussion
Functional evidence indicates that BeWo, Jeg-3 and JAr cells are able to take up organic anions via mechanisms sensitive to temperature and to self-inhibition by the substrate, suggesting that this process would be mediated by plasma membrane transporters. Here, four isoforms of the OATP family were studied as potential candidates to account for E217βG uptake by hCC. These transporters are able to mediate the uptake of a large variety of substrates, including bile acids and biliary pigments [16]
Acknowledgements
Secretarial help by M.I. Hernandez, technical help by E. Flores, assistance with immunohistochemical studies by M.T. Sanchez Montero, and supervision of the English version of the submitted manuscript by N. Skinner are gratefully acknowledged.
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Financial support: This study was supported in part by the “Junta de Castilla y León (Grant SA013/04)”, Spain and the “Ministerio de Ciencia y Tecnología, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Grant BFI2003-03208)”, Spain. Some of the authors are members of the Network for Cooperative Research on Hepatitis, Instituto de Salud Carlos III, FIS (Grant G03/015), Spain.