Reproductive Sciences

http://rsx.sagepub.com/ Autophagy-Related Proteins, LC3 and Beclin-1, in Placentas From Pregnancies Complicated by Preeclampsia
Soo-Young Oh, Suk-Joo Choi, Kyung Hee Kim, EunYoon Cho, Jong-Hwa Kim and Cheong-Rae Roh Reproductive Sciences 2008 15: 912 DOI: 10.1177/1933719108319159 The online version of this article can be found at: http://rsx.sagepub.com/content/15/9/912

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Autophagy-Related Proteins, LC3 and Beclin-1, in Placentas From Pregnancies Complicated by Preeclampsia
Soo-Young Oh, MD, Suk-Joo Choi, MD, Kyung Hee Kim, Eun Yoon Cho, MD, Jong-Hwa Kim, MD, and Cheong-Rae Roh, MD
The objective of this study is to investigate the expression of autophagy-related proteins (LC3 and beclin-1) in human placentas and the changes they undergo in the placentas from pregnancies complicated by preeclampsia (PE) and to explore the regulatory mechanisms of these proteins in JEG-3 cells in response to hypoxia or cytokine treatment.The presence of autophagosomes was confirmed with electron microscopy and the expression of LC3 and beclin-1 by immunoimaging methods in human placental trophoblasts. Compared with the placentas from normal pregnancies, the expression of LC3-II protein, but not beclin-1, was increased in the placentas from severe PE. In JEG-3 cells, hypoxia (O2 < 1%) induced a modest but not significant increase in the expression of LC3-II with a significant decrease in the expression of beclin-1. Meanwhile,TNF- treatment induced a significant increase in the expression of LC3-II without a significant change in the expression of beclin-1. Our data suggests that increased autophagic activity in the placenta may be implicated in the pathophysiology of PE. KEY WORDS: Placenta, autophagy, preeclampsia, beclin-1, LC3.

reeclampsia (PE) is one of the most frequent pregnancy complications and accounts for significant perinatal mortality and morbidity. Although the underlying mechanism of PE is not clearly understood, abnormal placentation and placental dysfunction are implicated in the etiology of this disorder. In addition, many investigators have reported increased trophoblast apoptosis in placentas of patients with PE1 suggesting that altered regulation of trophoblast apoptosis may contribute to the underlying pathophysiology of PE.2,3 However, mechanisms other than apoptosis may be associated with

From the Department of Obstetrics and Gynecology (SYO, SJC, KHK, JHK, CRR) and Department of Pathology (EYC), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea. This study was supported by Korea Research Foundation grants 2006-0470000 and 2007-0455-000. Presented in part at the 54th Annual Meeting of the Society for Gynecologic Investigation in Reno, Nevada, March 14-17, 2007 (abstract # 209). Address correspondence to: Cheong-Rae Roh, MD, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710, South Korea. E-mail: crroh@skku.edu.

Reproductive Sciences Vol. 15 No. 9 November 2008 912-920 DOI. 10.1177/1933719108319159 2008 by the Society for Gynecologic Investigation

the placental dysfunction associated with PE. Moreover, there is substantial evidence to support the fact that the placentas from women with PE are characterized by an exaggerated inflammatory response and oxidative stress compared with placentas from normal pregnancies.4 Autophagy, also called type II programmed cell death, is an intracellular bulk degradation system responsible for lysosomal degradation of protein and other subcellular constituents. It is often activated in response to nutritional deprivation and intracellular stress.5 Autophagy is an evolutionally conserved process that occurs in all eukaryotic cells, from yeast to mammals.6 Autophagy has been implicated in various physiologic processes including cellular differentiation and cell death. Moreover, autophagy and apoptosis are often coactivated in response to stress.7 In the past decade, rapid progress in the understanding of the molecular mechanism of autophagy has been made using genetic techniques for the study of yeast Saccharomyces cerevisiae.8 More recently, several mammalian homologues of yeast autophagy (ATG) genes have been identified. Among these, LC3, a mammalian homologue of yeast Apg 8/Aut7, was the first protein identified from the autophagosome membrane.9 Three isoforms of LC3 mRNA are currently known, LC3A, LC3B, and LC3C.10 After synthesis, LC3 is subsequently processed to LC3-I

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(cytosolic form) and modified to the active LC3-II form (membrane bound form) by posttranslational modification. LC3-II, after conjugation and lipidation with phosphatidylethanolamine, binds to the outer membrane of autophagosomes. Therefore, LC3 is a reliable marker of autophagosome formation in mammalian cells, and the relative amount of LC3-II reflects autophagic activity.11 Another important homologue of the yeast Apg6/Vps 30 is beclin-1, which is known to induce autophagy in response to nitrogen deprivation.12 Initially, beclin-1 was identified as the Bcl-2 interacting gene product13 necessary for nonapoptotic cell death.14 In contrast to LC3, a marker of final autophagosome formation, beclin-1 participates in the early stages of autophagy, promoting the nucleation of the autophagic vesicle and recruiting proteins from the cytosol.15 It has been reported that beclin-1 is monoallelically deleted in many human malignancies including breast cancer.12 Consistent with its involvement in the autophagic pathway, recent evidence has shown that beclin-1Bcl-2 interaction plays an essential role in cross-talk between apoptosis and autophagy in response to stress signals in mammalian cells.16 Excessive cytokine stress as well as hypoxia are commonly attributed to have some role in the pathophysiology of PE17 and these conditions are well known to be associated with an aberrant in utero environment and trophoblast cell kinetics.18 We hypothesized that the hypoxic microenvironment and increased cytokine stress in the placentas of patients with PE may induce autophagic activity in the placenta that contributes to the pathophysiology of PE.We investigated the expression of autophagy-related proteins (LC3 and beclin-1) in human placentas and their change in placentas from pregnancies complicated by severe PE. To explore the regulatory mechanism of these autophagy-related proteins in trophoblasts, we further analyzed the change of these proteins in response to hypoxia or cytokine treatment using the JEG-3 cell culture.

diagnosis of severe PE was based on the following. A blood pressure of 160/110 mm Hg or higher on 2 occasions at least 6 hours apart, proteinuria ( 4 g in a 24hour urine specimen or 2+ on 2 random dipsticks), accompanied by the presence of headache, visual disturbances, epigastric, right upper-quadrant pain, oliguria (<500 mL in 24 hours), pulmonary edema, impaired liver function, thrombocytopenia, or intrauterine growth restriction. After collection of the samples from the central mid-portion between the basal plate and chorionic membrane as described by Wyatt et al,19 the placental tissues were snap-frozen in liquid nitrogen and stored at 80C or embedded in paraffin for immunostaining. To investigate the changes in the autophagy-related proteins during advancing gestational age, the placentas from abortion or preterm births (n = 16) were also obtained. Any cases with multiple pregnancy, gestational diabetes, hypertension, and histologic chorioamnionitis were excluded from the study population.

Electron Microscopy
The placental tissues were fixed for 12 hours in 2.5% glutaraldehyde, washed in 0.1 M phosphate buffer (pH 7.4) twice for 15 minutes, and fixed again in aqueous 1% osmium tetroxide for 1 hour. After fixation, they were dehydrated in ethanol, embedded in Epok812, and incubated in an oven overnight at 37C. The tissue was then cut (50-60 nm) and stained with uranyl acetate for 30 minutes and lead citrate for 10 minutes. Electron microscopic examination was performed using a Hitachi 7100 transmission electron microscope (Japan).

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Real-Time Quantitative PCR
Total RNA was extracted from the placental tissue using Trizol (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturers instructions. One microgram of total RNA was used for reverse transcription with random hexamers using SuperScript III RT (Invitrogen). The following conditions

MATERIAL AND METHODS

Study Population and Tissue Collection
The Institutional Review Board for Clinical Research at Samsung Medical Center approved this study and an informed consent was obtained from each participant. Placentas were collected after cesarean delivery, without labor, from women with a normal term pregnancy (n = 8) as well as from women with severe PE (n = 11). The

were used for PCR: initial denaturation at 94C for 5 minutes, followed by 30 to 35 cycles of denaturation at 94C for 30 seconds, annealing at 55C for 30 seconds, and extension at 68C for 10 minutes using Accuprime Taq DNA polymerase (Invitrogen).The primers (National Center for Biotechnology Information accession number

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and product size) used for human LC3 genes were 5-CAGCATGGTGAGTGTGTCCA-3 (forward) and 5-GCAGCTCAGTTCAGGAACCA-3 (reverse) for LC3A (NM_181509, 195 bp), 5-GAGAGCAGCATCCAACCAAA-3 (forward) and 5-TCCGTTCACCAACAGGAAGA-3 (reverse) for LC3B (NM_022818, 185 bp), 5-GCAATCAGACAAGAGGAAGT-3 (forward) and 5-TAGAGAGGATTGCAGGGTC-3 (reverse) for LC3C (NM_001004343, 386 bp), and 5-GGCGGACTATGACTTAGTTG-3 (forward) and 5-AAACAACAATGTGCAATCAA-3 (reverse) for -actin (238 bp). All results were normalized to -actin to confirm a uniform amount of RNA template. For real-time quantitative PCR, the ABI Prism 7000 Sequence Detection System (PE Applied Biosystems, Foster City, CA) was used with the SYBR green detection system as described previously.20 PCR was performed in triplicate with 25 L reaction volumes of 5 L cDNA, 12.5 L SYBR Green PCR Master Mix, and 0.3 mmol/L of each primer. PCR was conducted using the following cycle parameters: 2 minutes at 50C, 10 minutes at 95C, 40 cycles of 15 seconds at 95C, and 1 minute at 60C. The primers (and product size) used for real-time quantitative PCR were 5-ATCATCGAGCGCTACAAGGGTG-3 (forward) and 5-TCATGTTGACATGGTCCGGG-3 (reverse) for LC3A (85 bp), 5-GCAAAACGCATTTGCCATCAC-3 (forward) and 5-CAGTTTACAGTCAGGGCCGTTTTC3 (reverse) for LC3B (80 bp), and 5-CGCCGCTAGAGGTGAAATTC-3 (forward) and 5-CGAACCTCCGACTTTCGTTCT-3 (reverse) for 18S rRNA (100bp). All results were normalized to 18S to ensure a uniform amount of RNA template.

assess the LC3 isoform expression in the placentas, we also used specific antibodies to LC3A and LC3B at 1:500 dilution for each (Novus Biologicals, Littleton, CO). After washing, the blots were incubated with anti-rabbit horseradish peroxidase conjugated secondary antibodies at a dilution of 1:5000 for 1 hour, and developed with ECL (Amersham Biosciences, Little Chalfont, UK). Immunostaining of LC3 was performed on paraffin-embedded sections using rabbit polyclonal antibody against LC3 (MBL, Nagoya, Japan). After deparaffination and

rehydration, the sections were incubated in 3% H2O2 in methanol for 30 minutes, and then microwaved. After blocking with 10% nonimmune serum, the sections were incubated with antibody to LC3 at a dilution of 1:1000 at 4C
overnight and followed by incubation with the biotinylated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at a dilution of 1:2000.After incubation in a streptavidinperoxidase conjugate, the antibody complexes were visualized with diaminobenzidine tetrahydrochloride chromogen for 10 seconds.The sections were counterstained with hematoxylin, dehydrated and mounted. Immunofluorescence microscopy of beclin-1 was performed on paraffin-embedded sections using rabbit polyclonal antibody to beclin-1 (Cell Signaling, Beverley, MA). The blocked sections were incubated with antibody to beclin-1 at a dilution of 1:1000 at 4C overnight and followed by incubation with the Alexa 594-rabbit IgG secondary antibody (Santa Cruz Biotechnology) for 1 hour at a dilution of 1:400 at room temperature. The sections were counterstained with DAPI, dehydrated, and mounted.

Culture of the JEG-3 Cell Line

The human choriocarcinoma JEG-3 cell line was obtained from the American Type Culture Collection (Rockville, MD). The cell line was maintained in RPMI 1640 medium (Gibco BRL, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum containing an antibiotic mixture at 37C under a humidified 5% CO2 atmosphere. About 1 105 cells per well, of a 6-well plate grown in RPMI 1640 medium, were supplemented with 10% fetal bovine serum and used for transfection.The culture medium was changed with 10% fetal bovine serum containing MEM medium about 4 hours before transfection and the cells were placed in a 10% CO2 incubator.The solution consisted of a half volume 2X BBS (N,N-bis[2hydroxyethyl]-2-aminoethanesulfonic acid-buffered saline, pH 6.95) (Sigma-Aldrich, St Louis, MO) and a half volume 0.25 M CaCl2 (Sigma-Aldrich).The 2X BBS solution was added to 5 g of GFP-LC3 plasmid DNA harboring LC3B (microtubuleassociated protein 1 light chain 3 beta, Map1lc3b, kindly

Western Blotting Analysis, Immunohistochemistry, and Immunofluorescence Staining

Proteins were extracted using RIPA buffer (50 mmol/L TrisCl, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS], pH 7.5) containing 1 mmol/L phenylmethylsulfonyl fluoride and quantified with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The protein samples (30 g) were separated by SDSpolyacrylamide gel electrophoresis (12% for LC3 and 8% for beclin-1) and then electrotransferred to a nitrocellulose membrane. After blocking with 5% nonfat dry milk in TBS-T (1 TBS with 0.1% Tween 20), the membranes were incubated with primary antibody (1:1000 for each) at 4C overnight.To

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provided by Dr Tamotsu Yoshimori, National Institute of Genetics, Mishima, Japan) and incubated for 30 minutes. The transfection mixture was then added to the cells under a 5% CO2 atmosphere at 37C. After 16 hours of transfection, cells were treated with 10 ng/mL tumor necrosis factor- (TNF-; Biosource International Inc, Camarillo, CA) or cultured in a

hypoxic chamber (Thermo Electron, Marietta, OH) providing hypoxic atmosphere defined as 1% O2 (5% CO2, 10% H2, and 85% N2) with pO2 < 15 mm Hg at 37C. The cell culture medium was changed every 24 hours.At a given time, the autophagic activity was assessed by observation of the punctuate GFP-LC3 staining using a confocal laser scanning microscope (CLSM, Radiance 2100 Rainbow, BioRad, Hercules, CA). Untransfected JEG-3 cells were also treated with 10 ng/mL TNF- or cultured in a hypoxic chamber for a given time and changes of LC3 and beclin-1 were assessed using immunoblot analysis.

Statistical Analysis
The MannWhitney U test and Fishers exact test were used to compare the clinical characteristics and differences between groups by mRNA and protein analysis. Spearman rank correlation test was performed to assess the changes of LC3 and beclin1 protein levels during advancing gestation. For data obtained from the JEG-3 cell culture, we used the KruskalWallis test to compare the protein levels of beclin-1 and LC3-II in 12, 24, and 48 hours in response to hypoxia or TNF- with reference level at time 0 point. The results were considered statistically significant when P values were less than .05.

RESULTS
The clinical characteristics of the participants are presented in Table 1. There was no difference in maternal age, maternal body mass index or neonatal gender. Multiparous women

were more common in the control group, reflecting the fact that most pregnant women in the control group had undergone an elective repeat cesarean delivery. As
expected, gestational age at delivery and neonatal birth weight were significantly lower in the severe PE group compared with the normal pregnancy group.

By electron microscopy, autophagic vacuoles were observed in both cytotrophoblast and syncytiotrophoblast of the human term placenta (Figure 1).These autophagic vacuoles contain intracytoplasmic organelles including mitochondria. The morphology of autophagic vacuoles

was somewhat various depending on the stage of autophagic vesicle formation and degradation of intravacuolar materials. First, to investigate whether there was any change in the level of LC3 and beclin-1 expression, in human placentas throughout gestation, placentas from various gestational ages after therapeutic abortion or preterm birth were assessed for these proteins. The human placentas expressed both LC3 and beclin-1 protein throughout gestation from the second trimester to term. There was no significant change of the levels of LC3 and beclin-1 in the placentas studied according to the gestational age (Figure 2). We evaluated the production of LC3 isoforms in the human placentas. Using RT-PCR, we observed the expression of LC3A mRNA and LC3B mRNA (Figure 3A) but could not detect LC3C mRNA in the human placentas (data not shown). Although the expression of LC3A mRNA in placentas was not different between the 2 groups, the expression of LC3B mRNA was significantly increased in the placentas from patients with PE compared with the normal pregnancies (Figure 3B).The Western blot analysis, using the specific antibody for LC3A and LC3B, confirmed the expression of these proteins in human placentas with dominant expression of LC3B (Figure 3C). Western blotting for LC3 showed increased levels of LC3-II expression in the placentas from patients with severe PE compared to patients with normal pregnancies, indicating increased autophagic activity in PE (Figure 4A). The immunohistochemistry results for LC3 also demonstrated more intense staining of the trophoblast layer in the placentas from patients with severe PE compared with the placentas from normal pregnancies (Figure 4B). We confirmed the expression of beclin-1 protein in human placentas. However, apart from LC3-II, there was no change in the expression of beclin-1 in the placentas from patients with severe PE compared with the normal pregnancies. Using immunofluorescence, beclin-1 was localized to the cytoplasm of the trophoblast layer in the human placentas (Figure 5). To determine the regulatory mechanism of these autophagy-related proteins in trophoblasts, we further analyzed the alteration of these proteins in response to hypoxia or cytokine treatment using the JEG-3 cell culture. The transfection study showed that the JEG-3 cells exposed to hypoxia or TNF- had a punctuate distribution of green fluorescence, indicating production of LC3 and its incorporation into autophagosomes (Figure 6A).

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Table 1. Clinical Characteristics of the Study Participants

Preterm (or Abortion) (n = 16) Maternal age (years)b Maternal BMI (kg/m2)b Multiparity (%) GA at delivery (weeks)b Birth weight (g)b Male neonate (%) 31 25.1 8 30.6 1463 10 (24-39) (20.3-29.7) (50.0) (14.4-34.4) (30-2458) (62.5) Normal (n = 8) 35 25.4 6 38.6 3265 5 (26-38) (24.0-35.0) (75.0) (38.3-39.3) (3010-4220) (62.5) Severe PE (n = 11) 33 28.3 3 33.1 1671 4 (25-38) (17.6-36.0) (27.3) (28.0-40.1) (740-3200) (36.4) P Valuea NS NS .07 <.05 <.001 NS

Abbreviations: PE, preeclampsia; BMI, body mass index; GA, gestational age; NS, nonsignificant. a Normal group versus severe PE group by MannWhitney U test or Fishers exact test. b Data are expressed as median (range).

Figure 1. Electron microscopy images of human placentas. Scanning of both cytotrophoblast (A) and syncytiotrophoblast (B) with low and high power fields.Arrows indicate the autophagic vacuoles.The shapes of autophagic vacuoles vary depending on the stage of autophagome formation. In the right panel, in a double membranous vesicle, cytoplasmic organelles including mitochondria are being lysed. In the left panel, autolysosome was formed with broken mitochondria suggesting later stage of autophagosome formation. Note that syncytiotrophoblast contains a vacuolized cytoplasm and more autophagosomes than cytotrophoblast. Scale bars, 1 m; scale bars of insets, 100 nm.

Figure 3. The expression of LC3A and LC3B in human placentas from normal pregnancies and those with preeclampsia: reverse transcriptase polymerase chain reaction, RT-PCR (A), real-time qPCR (B), and immunoblotting (C). The expression of LC3B mRNA, but not LC3A mRNA, was significantly increased in placentas with severe PE compared with normal pregnancies (data are presented as mean standard error of the mean). The Western blot analysis, using the specific antibody for LC3A and LC3B, confirmed the expression of these proteins in human placentas with dominant expression of LC3B. Note that the unprocessed form of LC3A protein was barely detected (C). Representative cases are shown for the RT-PCR and Western blot of LC3A and LC3B. *P < .05.

Figure 2. The expression of LC3 and beclin-1 in human placentas throughout gestation. By Western blot, it was found that the human placentas expressed both LC3 and beclin-1 at all gestational ages studied. After densitometric analysis and normalization to -actin, it was concluded that there were no significant changes in the protein levels of LC3 and beclin-1 according to the gestational age from second trimester to term pregnancy (P = nonsignificant, by Spearman rank correlation test). Representative blots are shown.

In addition, we found that the untransfected JEG-3 cells exposed to hypoxia or TNF- showed a distinct change of LC3 and beclin-1 proteins in a given time course. In JEG-3 cells, hypoxia (O2 < 1%) induced a modest increase in the expression of LC3-II (without a statistical significance, P = .15) with a significant decrease in the expression of beclin-1 (P < .05). Meanwhile, TNF-

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Figure 4. The expression of LC3 in human placentas from normal pregnancy and preeclampsia. A, Immunoblotting analysis shows that the level of LC3-II expression was significantly increased in severe PE compared with normal pregnancies.The data are presented as mean SEM. Representative blots are shown. *P < .05. B, Immunostaining for LC3 in placental villi. (a) No primary antibody control; (b) Negative control with primary antibody preabsorbed with blocking peptides; (c) Normal term placenta; and (d) Placenta with preeclampsia. LC3 immunostaining was predominant in the cytoplasm of the trophoblasts. Note the marked difference in LC3 expression in placentas from PE compared with normal pregnancies (left panel, original magnification 400; bar = 15 m; right panel, original magnification 1000).

Figure 6. The expression of LC3 and beclin-1 in JEG-3 cells after treatment with hypoxia and TNF-. A, GFP-LC3 fluorescence in JEG-3 cells. JEG-3 cells were transfected with GFP-LC3 and then cultured in a hypoxic chamber (O2 < 1%) or treated with 10 ng/mL TNF-.Whereas JEG-3 cells transfected with empty plasmid did not show any distribution of GFP-LC3, the cells transfected with GFP-LC3 plasmid showed more and stronger green fluorescent punctuations after 24 hours in response to hypoxia or TNF- compared with null, indicating more production of LC3 and its incorporation into autophagosomes. B, Western blot analysis of LC3 in JEG-3 cells in response to hypoxia or TNF-. Although hypoxia induced a modest increase in the expression of LC3-II (P = .15, KruskalWallis test),TNF- induced a significant increase in the expression of LC3-II in 48 hours of treatment (*P < .05, KruskalWallis test). C,Western blot analysis of beclin-1 in JEG-3 cells in response to hypoxia or TNF-.The expression of beclin-1 in cells was significantly decreased during 12 to 48 hours of culture in a hypoxic chamber (*P < .05, KruskalWallis test). TNF- did not induce a significant change in the expression of beclin-1 at a given time. Similar results were obtained in 3 independent experiments.The data are presented as mean SEM.

Figure 5. The expression of beclin-1 in human placentas from normal pregnancy and preeclampsia. A, Immunoblotting analysis showed that the level of beclin-1 expression in the placenta from preeclampsia was similar to normal pregnancies.The data are presented as mean SEM. Representative blots are shown. B, Immunofluorescence staining shows beclin-1 (red) was diffusely localized to the cytoplasm of the trophoblast cell layer in human placentas (original magnification 400).

treatment induced a significant increase in the expression of LC3-II (P < .05) without a significant change in the expression of beclin-1 (Figure 6B and C).

COMMENT
To our knowledge, this is the first study to identify autophagosomes and autophagy-related proteins (LC3 and beclin-1) in human placentas and to demonstrate

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their altered expression in association with PE. We also showed modification of these proteins in a trophoblast cell line in response to hypoxia or cytokine treatment. Autophagy is a dynamic process that allows cells to remove damaged organelles and recycle amnio acids.The standard method of assessing autophagy is by direct visualization of the autophagosomes using an electron microscope. Morphologically, we observed that the trophoblast cytoplasm constituents including the mitochondria were sequestered into the double membrane of the autophagosomes, which subsequently fused with lysosomes and then their contents were degraded. However, electron microscopy is a timeconsuming procedure and is subject to biased interpretation.21 Therefore, immunoblotting of LC3-II is preferred as a simple and quantitative method for specifically determining autophagic activity in mammalian cells.9,21 Our data showed that the level of LC3B mRNA and the protein expression of LC3-II were increased in the placentas from patients with severe PE compared with normal pregnancies, suggesting increased autophagic activity in PE. The inherent limitation of this study may be the fact that gestational age of placenta collection was different between normal and PE groups. Because most of preterm placentas are obtained after preterm labor or preterm premature rupture of membrane, in which underlying inflammation may exist, it is hard to obtain normal preterm control placentas.Therefore, we evaluated the expression of LC3 as well as beclin-1 in placentas from patients with severe PE in third trimester of pregnancy and used term placentas as a control group.The second limitation of our study lies that the relative small number of placentas for study of the gestational age-associated change.Therefore, it seems to be more prudent to include more normal preterm placentas in the future to strengthen this in vivo result and to reach any conclusions. LC3 was originally copurified with the microtubuleassociated protein 1A and IB from rat brain.22 It has a 28% amino acid sequence homology with the yeast Apg 8/Aut7, which is essential for yeast autophagy.12 LC3 was the first protein identified on the autophagosome membrane.9 Three types of LC3 sequences have been identified to date, LC3A, LC3B, and LC3C.10 Based on our study results, the mRNA expression of LC3 was confined to LC3A and LC3B, but not to LC3C. After synthesis, LC3 undergoes ubiquitin-like posttranslational modifications before autophagosome formation. Immediately after synthesis, the C-terminal region of LC3 is cleaved to form LC3-I, which has a glycine residue at the C-terminal

end. LC3-I is located in the cytosol; it is further processed and incorporated into the autophagosome membrane as LC3-II.9 Therefore, LC3-II is specifically associated with autophagosome formation. Our data showed that the level of unprocessed LC3 in the human placentas was relatively low (Figure 3C) compared with those of LC3-I and LC3-II (Figure 4A). This finding suggests a rapid processing of LC3 especially in PE after its initial synthesis (Figure 3B). The most common inducer of LC3-II is nutrition deprivation, but other cellular stress such as hypoxia can also trigger autophagy.23 Moreover, normal cellular differentiation is associated with increased LC3II. Of note is that LC3-II is increased during the differentiation of murine podocyte cells.24 The results of our study showed that hypoxia or cytokine treatment increased the expression of LC3-II in the trophoblast cell line. In fact, hypoxia inhibits the phosphorylation of the mTOR (mammalian target of rapamycin) pathway, which is an important upstream signaling pathway that negatively regulates autophagy.25 Beclin-1 is an important regulator of autophagy. Expression of beclin-1 in MCF7 mammalian cells increases their autophagic response to nutrient deprivation.12 Suppression of beclin-1 in mammalian cells impairs autophagy and sensitizes the cells to starvation-induced autophagy.26 In our study, we initially speculated that the expression of beclin-1 in the placentas from women with PE would be increased when compared with normal placentas. However, unlike the LC3-II protein, we found that there was no change in the level of placental beclin1 expression in the severe PE placentas compared with the normal placentas. To explore the underlying mechanisms to explain our ex vivo data, we performed in vitro experiments using a trophoblast cell line and demonstrated significant changes in the expression of LC3-II and beclin-1 depending on the type of stimulation (hypoxia or TNF-). Our data showed that TNF- rather than hypoxia provoked a significant up-regulation of LC3-II in trophoblast cell line. On the contrary, hypoxia rather than TNF- induced a significant downregulation of beclin-1. The effect of hypoxia on the expression of LC3-II and beclin-1 was similar after treatment of JEG-3 cells with CoCl2 (data not shown). Although beclin-1 is known to induce autophagy, it is debated whether the expression of beclin-1 increases the autophagic response. For example,Yan et al23 demonstrated that the expression of beclin-1, along with LC3-II, was increased when the chronically ischemic myocardium of

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pigs induced autophagy, whereas Martinet et al21 found that starvation-induced autophagy was not associated with changes in the expression of beclin-1 in HepG2 cells. Moreover, Tsujimoto et al27 proposed that withdrawal of amino acids or lymphokine stimulation maintained a low level of atg5/atg6 during autophagy for survival; this is consistent with our in vitro data. Therefore, the increased autophagic activity in the placentas from women with PE might reflect a trophoblast survival response to excessive inflammatory stimuli and oxidative stress in the PE placentas. However, in our data, there remains a question why the expression of beclin-1 decrease in JEG-3 cells in response to hypoxia although the expression of beclin-1 in placentas was not different between normal and PE pregnancies. We are not sure exactly why this discrepancy occurs. Nevertheless, this may be partly attributable to the difference between in vivo oxygen milieu and our in vitro experimental condition. It has been suggested by other researchers that there might be spatial and temporal variations of oxygen tension within the intervillous space in a disturbed PE placenta.28 In summary, we demonstrated that the expression of LC3, but not beclin-1, was increased in placentas from pregnancies complicated by severe PE compared with normal placentas suggesting increased autophagic activity in placentas from patients with PE. In addition, our in vitro findings showed that this autophagic response could be regulated by trophoblast microenvironment (ie, hypoxia or cytokine). Taken together, the altered expression of these autophagy-related proteins in PE may be implicated in the pathophysiology of this disorder and this research agenda warrants future intense investigations.

ACKNOWLEDGMENTS
We are grateful to Dr Yoel Sadovsky (Scientific Director,

Magee-Womens Research Institute & Foundation, Pittsburgh, PA) for helpful discussion during this investigation.

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