Isolation of basal membrane proteins from BeWo cells and their expression in placentas from fetal growth-restricted pregnancies☆
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.
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This work was partially presented at 2013 IFPA meeting held in Whistler, Canada.
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Soo-young Oh and Jae Ryoung Hwang contributed equally to this work as the first authors.