Elsevier

Placenta

Volume 39, March 2016, Pages 24-32
Placenta

Isolation of basal membrane proteins from BeWo cells and their expression in placentas from fetal growth-restricted pregnancies

https://doi.org/10.1016/j.placenta.2016.01.001Get rights and content

Highlights

  • We isolated the basal membrane from BeWo cells using the cationic colloidal silica method.

  • HO-1, VDAC1, and RPN2 proteins were identified as basal membrane proteins.

  • Hypoxia increased the membranous localization of HO-1, VDAC1, and RPN2 proteins.

  • Truncated HO-1 was expressed in the basal membrane fraction of human placenta.

  • FGR significantly reduced the expression of truncated HO-1.

Abstract

Introduction

The syncytiotrophoblast, a key barrier between the mother and fetus, is a polarized epithelium composed of a microvillus and basal membrane (BM). We sought to characterize BM proteins of BeWo cells in relation to hypoxia and to investigate their expression in placentas from pregnancies complicated by fetal growth restriction (FGR).

Methods

We isolated the BM fraction of BeWo cells by the cationic colloidal silica method and identified proteins enriched in this fraction by mass spectrometry. We evaluated the effect of hypoxia on the expression and intracellular localization of identified proteins and compared their expression in BM fractions of FGR placentas to those from normal pregnancies.

Results

We identified BM proteins from BeWo cells. Among BM proteins, we further characterized heme oxygenase-1 (HO-1), voltage-dependent anion channel-1 (VDAC1), and ribophorin II (RPN2), based on their relevance to placental biology. Hypoxia enhanced the localization of these proteins to the BM of BeWo cells. HO-1, VDAC1, and RPN2 were selectively expressed in the human placental BM fraction. C-terminally truncated HO-1 was identified in placental BM fractions, and its BM expression was significantly reduced in FGR placentas than in normal placentas. Interestingly, a truncated HO-1 construct was predominantly localized in the BM in response to hypoxia and co-localized with VDAC1 in BeWo cells.

Discussion

Hypoxia increased the BM localization of HO-1, VDAC1, and RPN2 proteins. FGR significantly reduced the expression of truncated HO-1, which was surmised to co-localize with VDAC1 in hypoxic BeWo cells.

Introduction

The human syncytiotrophoblast layer is a polarized epithelium composed of an apical microvillus membrane (MVM) and a basal membrane (BM), which plays a key role in regulating maternal–fetal communication. Diverse trophoblastic proteins are differentially expressed in maternal-facing MVM or fetal-facing BM [1], [2]. Selected transports from mother to fetus are modulated by the trophoblastic BM. Examples of such transports include transplacental glucose transfer and important feedback that controls placental iron transport [3], [4]. Although BM function is critical, our current understanding of this function is inadequate. Functional studies are hampered, in part, by the technical difficulty in isolating the trophoblastic BM [5]. Whereas proteomic analysis of the human syncytiotrophoblast has identified enrichment of specific proteins, such as annexin A2 and alkaline phosphatase, in MVM lipid rafts [6], proteomic analysis of the human trophoblastic BM has not been hitherto reported.

The cationic colloidal silica (CCS) method, first described by Chaney and Jacobson in the early 1980s to isolate plasma membranes from suspended cells, has undergone many modifications and has been applied for purifying the luminal endothelial cell membrane and basolateral membrane of adherent cells [7]. Recently, the efficiency of the CCS method for capturing the basolateral integral membrane in a colon adenocarcinoma cell line was validated, revealing minimal contamination from other membranes and basic proteins [8]. The trophoblastic BeWo cell line was identified as a polar cell model and thus was suitable for the study of MVM and BM fractions [3], [9].

Using the CCS method, we here isolated the BM of BeWo cells and performed a proteomic analysis to identify proteins enriched in the BM. Among proteins enriched in the BM fraction as observed by mass spectrometry, we focused three proteins, HO-1, VDAC1, and RPN2, based on their relevance to maternal–fetal communication. We investigated the expression of these proteins in BeWo cells cultured in standard or hypoxic conditions and correlated their expression patterns in normal placentas or in placentas from pregnancies complicated by fetal growth restriction (FGR). We found that truncated HO-1 was constitutionally expressed in the BM fraction of human placenta, and we further studied the change in expression and intracellular localization of truncated HO-1 in response to hypoxia by using a truncated HO-1 construct (Flag-HO-1△C23), which has deletion in the C-terminal region of HO-1.

Section snippets

Cell culture

BeWo cells were obtained from Korean Cell Line Bank (KCLB No. 10098) and were maintained in Ham's F-12K (Kaighn's) Medium (Invitrogen, Life Technologies, USA) containing 10% fetal bovine serum and penicillin/streptomycin (Invitrogen). To provide a hypoxic environment, we used a hypoxia incubator chamber (1% O2; Forma Anaerobic System).

Isolation of the BM fraction from BeWo cells

The BM fraction was isolated on the basis of a previously described method [8]. Briefly, BeWo cells were washed with cold PBS (pH 7.4) containing 1 mM MgCl2 and

Isolation of the BM fraction of BeWo cells by using the CCS method

We isolated the BM fraction of BeWo cells by using the CCS method and confirmed the fractionation efficiency by detecting marker proteins that characterize each fraction (Fig. 1A). Unlike the scant expression in whole cell lysates, the expression of PMCA and adenyl cyclase (markers of BM) was highly enriched in BM fractions. In contrast, PLAP (a marker of MVM) was not detected in BM fractions. Cytochrome C was detected only in whole cell lysates, demonstrating no mitochondrial contamination of

Discussion

Several methods have been used to isolate the trophoblastic BM from human placenta. One approach used was sonication and incubation with EDTA to selectively isolate the BM followed by centrifugation [13] and further purification through a Ficoll or sucrose gradient [14]. Another method for isolating MVM and BM from trophoblasts, reported by Illsley et al. [15], involved homogenization, Mg2+ precipitation, and purification by sucrose gradient. We used this isolation procedure to prepare MVM and

Role of funding source

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Education (NRF-2012R1A1A3006404) and by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0024 (A120035)).

Acknowledgments

We are grateful to Professor Theresa L. Powell (Section of Neonatology, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA) for kindly providing the protocol for preparation of membrane fractions from placental tissues.

References (36)

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    In addition, HMGB3 may also interact with voltage-dependent anion channel-1 (VDAC1). Hypoxia induced an increase in the expression of VDAC1 and heme oxygenase-1 and C-terminally truncated heme oxygenase-1 was significantly decreased in FGR placenta (Oh et al., 2016). VDAC1 and heme oxygenase-1 appear to be co-localized in the basement membrane of syncytiotrophoblastic cells (Oh et al., 2016), suggesting that changes of VDAC1 expression induced by hypoxia may play a role in the pathogenesis of FGR.

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This work was partially presented at 2013 IFPA meeting held in Whistler, Canada.

1

Soo-young Oh and Jae Ryoung Hwang contributed equally to this work as the first authors.

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