Effects of maturation on RNA transcription and protein expression of four MRP genes in human placenta and in BeWo cells

https://doi.org/10.1016/S0006-291X(03)00327-9Get rights and content

Abstract

The placenta is a multifunctional organ that protects the fetus from toxic compounds and the MRPs contribute to this function. The expression of MRP1, MRP2, MRP3, and MRP5 was compared in human placental tissue and in BeWo cells by real-time RT-PCR analysis; protein expression was assessed by Western blot. MRP1 and MRP3 were the most abundantly expressed genes in placenta but only MRP1 was highly expressed in the BeWo cells. Expression of MRP1 increased 4-fold in the third as compared with first trimester placental samples, and increased 20-fold with polarization of BeWo cells. MRP2, MRP3, and MRP5 were weakly expressed both in placenta and BeWo cells. Protein expression followed mRNA quantification for MRP1 and MRP5 but not for MRP2 and MRP3. These data indicated that MRP1 and MRP5 increase with trophoblast maturation, suggesting a particular role for these proteins in the organ functional development.

Section snippets

Materials and methods

Placental tissues and cell cultures. Normal human placentas were obtained at term from healthy pregnancies and in the first trimester (9–10 weeks of gestation) from therapeutic interruptions of pregnancy. These were kindly provided by Gynecology and Obstetric Department of “Ospedale Infantile Burlo Garofolo,” Trieste, Italy, with a protocol approved by the Ethical Committee of the University of Trieste.

BeWo cells were obtained from Istituto Zooprofilattico Sperimentale (Brescia, Italy) and cell

Quantitative PCR

The melting dissociation profiles (data not shown), performed on MRP1, MRP2, MRP3, and MRP5 cDNAs, allowed confirmation of the specificity of the amplifications. The melting temperatures of MRP1, MRP2, MRP3, and MRP5 amplicons were 86.9±0.2, 86.1±0.1, 83.3±0.4, and 84.7±0.5°C, respectively, whereas GAPDH had a melting temperature of 81.9±0.2°C. The specificity of the amplification was also confirmed by direct sequencing and sequence alignment with the MRP1, MRP2, MRP3, and MRP5 sequences

Discussion

During gestation, it is essential that the development of the placenta is synchronized with that of the embryo. The placental barrier, which interfaces between the fetus and the mother, must cope with the gradually increasing requirements of the fetus for supply of nutrients and removal of toxic metabolites. At the different stages of fetal development, these functions must be served by a constantly evolving differential expression of the various genes involved in the relevant transplacental

Acknowledgements

Authors are deeply grateful to Dr. J.D. Ostrow for critical reading of the manuscript. L.P. and C.F. were supported in part by career development awards from Bracco SpA, Italy; D.P. was supported by a long term fellowship from University of Trieste. This work was also supported by IRCCS Burlo Garofolo Grant RF98/67, by grants from the Italian Ministry of Education (MIUR, Rome, Italy) and Fondo Studi Fegato of Trieste, Italy (FCRT-00-02).

References (36)

  • M.A. Serrano et al.

    Evidence for carrier-mediated transport of unconjugated bilirubin across plasma membrane vesicles from human placental trophoblast

    Placenta

    (2002)
  • G.L. Scheffer et al.

    Tissue distribution and induction of human multidrug resistant protein 3

    Lab. Invest.

    (2002)
  • G. Jedlitschky et al.

    The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides

    J. Biol. Chem.

    (2000)
  • K. Lee et al.

    Fetal bilirubin metabolism and neonatal jaundice

  • H. Schneider

    Placental transport function

    Reprod. Fertil. Dev.

    (1991)
  • D.P. Faller et al.

    Evidence for location of the CFTR in human placental apical membrane vesicles

    Am. J. Physiol.

    (1995)
  • A.J. Moe

    Placental amino acid transport

    Am. J. Physiol.

    (1995)
  • P. Bravo et al.

    Interaction between cholephilic anions and bile acid transport across basal membrane of human trophoblast

    Am. J. Physiol.

    (1993)
  • Cited by (85)

    • Role of the efflux transporters BCRP and MRP1 in human placental bio-disposition of pravastatin

      2018, Biochemical Pharmacology
      Citation Excerpt :

      Thus, the results of inhibition experiments with 100 µM of indomethacin and benzbromarone (Fig. 4) showed that the uptake of [3H]-pravastatin determined in placental vesicles prepared from apical and basal membranes is mediated by multiple efflux transporters. Taking into consideration results from western blot analysis and previously reported high mRNA expression of BCRP [34,35] especially in term placentas [11] as well as relative abundance of MRP1 compared to other MRPs [34,36], the in vitro ATP-dependent uptake of [3H]-pravastatin by membrane vesicles could be primarily attributed to BCRP and MRP1. In the range of pravastatin concentrations tested, uptake of [3H]-pravastatin by BCRP or MRP1 overexpressed vesicles showed standard saturation curves with relatively high affinity to the transporters (Fig. 5B&D).

    • Don't trust an(t)ybody - Pitfalls during investigation of candidate proteins for methylmercury transport at the placental interface

      2016, Placenta
      Citation Excerpt :

      High MRP2 levels in human placentas were shown in four studies (using monoclonal antibody clone M2III-6 [20–22] and antibody clone M2 I-4 [23]). Our data, however, are in line with two other studies, where MRP2 expression could either not be detected in crude placental membrane fractions and BeWo cells (M2III-6 [24]) or only very faint bands were seen in trophoblast membrane fractions and BeWo cells (M2 I-4; [25]). None of these studies had tested antibody specificity by e.g., protein overexpression or downregulation as recently suggested [10], and in all studies only parts of immunoblots were shown.

    View all citing articles on Scopus
    View full text