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. 2012 Aug 9;153(10):4946–4954. doi: 10.1210/en.2012-1472

Hypoxia Induces Autophagy in Primary Human Trophoblasts

Baosheng Chen 1,✉, Mark S Longtine 1, D Michael Nelson 1
PMCID: PMC3512007  PMID: 22878401

Abstract

Autophagy is a highly regulated and dynamic process that maintains cellular homeostasis and plays a prosurvival role in most cells. Although hypoxia has been shown to induce apoptosis in placental trophoblasts, the hypoxic effect on autophagy has not been studied. We hypothesized that autophagy plays a prosurvival role in the placental trophoblasts by antagonizing hypoxia-induced apoptosis. Our data show that the expression of Light chain 3-II (LC3-II), an autophagic marker and cleaved poly(ADP-ribose) polymerase, an apoptosis marker, are inversely related in cultured trophoblasts. Exposure to rapamycin or hypoxia inactivated mammalian target of rapamycin, as reflected by reduced phosphorylation of ribosomal protein S6, indicating that mammalian target of rapamycin regulates autophagy in cultured cytotrophoblasts. Bafilomycin prevented the degradation of cargo and increased LC3-II and p62 in cytotrophoblasts exposed to hypoxia, revealing enhanced autophagic flux. Importantly, bafilomycin enhanced expression of autophagy-related protein 7 (Atg7), parallel to the increased apoptosis measured by cleaved poly(ADP-ribose) polymerase. LY294002, a phosphatidylinositol 3-kinase inhibitor, increased apoptosis in the trophoblasts under hypoxia or standard conditions. Silencing of Atg7 decreased both apoptosis and LC3-II in the trophoblasts, suggesting a dual role of Atg7 in both autophagy and apoptosis. We conclude that there is a cross talk between autophagy and apoptosis in the placental trophoblasts; autophagy plays a prosurvival role and Atg7 has roles in both autophagy and apoptosis under hypoxia.

The human placenta is a transient organ that connects the developing fetus to the maternal uterine wall. The placenta provides endocrine and immunological functions and is the site of uptake of nutrients, gas exchange, and waste removal for the fetus. The chorionic villi of human placentas are bathed by maternal blood and surfaced by an epithelial bilayer of trophoblasts. One layer is the syncytiotrophoblast, which is terminally differentiated and lacks lateral cell membranes to partition individual nuclei and organelles. An adjacent discontinuous layer of cytotrophoblasts can divide and fuse with the syncytiotrophoblast, serving as a stem cell population for villous growth and repair. During pregnancy, the villous trophoblast bilayer is exposed to stresses created by variable maternal blood flow in the intervillous space, which creates oxidative stress from hypoxia and/or reoxygenation. Both villous trophoblast layers are reported to undergo apoptosis (1, 2), but in the second half of pregnancy cytotrophoblasts are more sensitive than syncytiotrophoblasts to hypoxic conditions in vivo (1) and in vitro (3, 4). Preeclampsia and intrauterine growth restriction, maladies that occur, individually or together, in greater than 10% of pregnancies (5), have been reported to be associated with placental dysfunction from injury due to oxidative stress. Furthermore, apoptosis in villous trophoblasts is higher than normal in such conditions (5–7). Given that widespread apoptosis in villi would be catastrophic to the placenta, and thus, to the developing fetus, we predicted that human villous trophoblasts display mechanisms to maintain homeostasis and protect from dysregulated apoptosis.

One mechanism by which cells protect themselves from stressors is autophagy, a catabolic process involving the capture of cellular constituents by double-membrane autophagosomes that fuse with lysosomes into autolysosomes in which the contents are degraded and released for reuse. Autophagy is important in development, tissue differentiation, and tissue remodeling (8), and the process occurs at a basal level in most cells to maintain homeostasis. Flux through the autophagic pathway is elevated in response to multiple stressors, including starvation, hypoxia, and excess reactive oxygen species. Notably, stress-induced increase in autophagy is typically cytoprotective, allowing cells to survive during the period of exposure (9).

Autophagy is regulated by mammalian target of rapamycin (mTOR), a kinase that integrates nutritional status and stress signals (10–13). Downstream of mTOR are a group of autophagy-related proteins (Atg) with distinct functions in each step of the autophagic process (Fig. 1A) (14). Atg8/Light chain 3-II (LC3-II) is the only Atg protein to persist on autophagosomes from their formation to degradation. Thus, LC3-II and p62, which is degraded during autophagy, are often used as markers for autophagy (15). Notably, Atg7 is important in forming the autophagosome by acting as an E1-like enzyme for two ubiquitin-like conjugation systems, in which Atg12 is conjugated to Atg5, and Atg8/LC3 is conjugated to phosphatidylethanolamine, forming LC3-II (16).

Fig. 1.

Fig. 1.

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A, Diagram of methods and autophagy pathway tested in the study. Rapamycin was used to inhibit mTOR kinase activity; bafilomycin, LY294002, and siRNA for Atg7 were used to inhibit different stages of autophagy pathway. Phosphorylated ribosomal protein S6 (p-S6) was used to measure mTOR activity. LC3-II and p62 were used as readouts for autophagy. B, The expression of LC3-II and cleaved PARP (cl-PARP) in the cultured primary human trophoblasts. Primary human trophoblasts were plated for 4 h, washed three times with PBS, and then continued in culture for 20 h (time here defined as 0, as labeled in the figure). The medium was then replaced, and cells were cultured for up to an additional 8 h. Left panel shows representative Western blots of LC3-II and cl-PARP. Middle and right panels show summary graphs of densitometry of Western blots of LC3-II and cl-PARP, respectively. In this and all following figures, the values of densitometry are normalized to actin used for the internal control. Data are presented as mean ±sd, with n = 3. *, P < 0.05 by one-way ANOVA/Bonferroni's post hoc test.

Recent evidence suggests that autophagy might play an important role in villous trophoblasts. Several autophagy-related proteins, including multiple components of mTOR complex (mTORC)-1, are expressed in human placental villous trophoblasts (17–19). Moreover, enhanced autophagy is reported in villi of pregnancies from women with preeclampsia, compared with normotensive pregnancies (20, 21). Despite this, our understanding of the regulation of autophagy in placental pathophysiology is very limited. Important questions to be resolved include how autophagy is regulated in trophoblasts and how regulators in the autophagy pathway interact with regulators of apoptosis to determine survival or demise in human villous trophoblasts exposed to stress. In the current study, we used in vitro primary cultures of human cytotrophoblasts exposed to standard or hypoxic culture conditions to test thehypothesis that term cytotrophoblasts undergo autophagy in response to hypoxic stress. Moreover, we built on our previous data showing that cytotrophoblasts also respond to hypoxia with enhanced apoptosis and tested the hypothesis that autophagy and apoptosis are inversely related in hypoxic cytotrophoblasts. We identified Atg7 as a key player in this dual regulation.

Materials and Methods

Isolation and culture of primary human trophoblasts

This study was approved by the Institutional Review Board of Washington University School of Medicine (St. Louis, MO). Primary human trophoblasts were isolated from uncomplicated singleton pregnancies delivered by repeat cesarean section at 39–40 wk gestation, as previously described (3, 22). Cultures were plated at a density of 300,000 cells/cm2 and maintained in DMEM (Sigma, St. Louis, MO) containing 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY), 20 mm HEPES (pH 7.4) (Sigma), penicillin (100 U/ml), streptomycin (100 μg/ml), and Fungizone (0.25 μg/ml; all from Washington University tissue culture support center) at 37 C in a 5% CO2-air atmosphere for 4 h to allow cell attachment. The cells were washed three times with PBS and then cultured in fresh medium for the desired time period for further experiments. For the data in Figs. 2–6, after 4 h to allow attachment, the cells were either grown in standard conditions of 5% CO2-air or exposed to hypoxia (<1% O2/5% CO2/10% H2/balance N2) for up to 24 h, with or without rapamycin [300 nm (23–25); Fisher Scientific, St. Peters, MO], bafilomycin [100 nm (23, 26–29); Alexis Biochemicals, San Diego, CA], LY294002 [10 μm (30–32), Cell Signaling Technology, Danvers, MA], or dimethylsulfoxide (DMSO) vehicle control, for the times noted in the figure legend. For the data in Figs. 1 and 7, the detailed times and treatments are listed in the corresponding figure legends. The less than 1% O2 environment was supplied in an anaerobic glove box incubator (Thermo Electron, Marietta, OH) that allowed pregassing of the medium and the handling of cultures without exposure to ambient conditions.

Fig. 2.

Fig. 2.

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The effect of rapamycin, bafilomycin, and hypoxia (<1% O2) on the expression of LC3-II and phosphorylated ribosomal protein S6. A, Western blots of LC3-II and phosphorylated ribosomal protein S6 in trophoblasts exposed to rapamycin, bafilomycin, or both. Trophoblasts were plated and cultured for 4 h in standard conditions (20% O2) and then exposed to rapamycin (300 nm) or bafilomycin (100 nm) for 4 h. Expression of phosphorylated ribosomal protein S6 was used as a marker for mTOR activity. B, Western blots of LC3-II and phosphorylated ribosomal protein S6 in trophoblasts exposed to hypoxia (<1% O2) compared with standard conditions (20% O2). Trophoblasts were plated and cultured for 4 h in standard conditions and then split with half cultured in hypoxia and the other half in standard conditions for up to 24 h. Representative Western blots are shown on the upper panel. The lower panel shows the summary graph of densitometry of LC3-II. Data are presented as mean ±sd, with n = 3. *, P < 0.05 by two-way ANOVA/Bonferroni's post hoc test.

Fig. 3.

Fig. 3.

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Hypoxia activates autophagic flux in cultured trophoblasts. Trophoblasts were plated and cultured for 4 h in standard conditions and then exposed to bafilomycin (100 nm) for up to 24 h in standard conditions (A) or hypoxic conditions (B). Upper panels of A and B show representative Western blots of LC3-II expression in trophoblasts. Lower panels show the summary graphs of the densitometry of LC3-II Western blots. Data are presented as mean ±sd, with n = 3. *, P < 0.05 by two-way ANOVA/Bonferroni's post hoc test. C, Autophagic flux analyses shown by expression changes of LC3-II in trophoblasts exposed to bafilomycin under hypoxic conditions compared with standard conditions. Data are presented as mean ±sd, with n = 3. *, P < 0.05 by two-way ANOVA/Bonferroni's post hoc test.

Fig. 4.

Fig. 4.

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The effect of hypoxia, bafilomycin, or both on the expression of p62 in trophoblasts. Trophoblasts were plated and cultured for 4 h under standard conditions and then exposed to hypoxia compared with standard conditions for 4 h either with bafilomycin (100 nm) or with DMSO control. The cells were fixed and probed for p62 using immunofluorescence staining. Representative pictures are shown for p62 staining under standard or hypoxic conditions, with or without bafilomycin. The signal of p62 was quantified by counting p62 bodies with a diameter greater than 0.5 μm in 10 randomly selected fields and normalized to the total nuclei in the selected fields. The result is presented in the graph at the bottom of the pictures. Data are presented as mean ±sd. *, P < 0.05 by two way ANOVA/Bonferroni's post hoc test.

Fig. 5.

Fig. 5.

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The effect of bafilomycin on the expression of cleaved PARP and Atg7 in trophoblasts. A, Trophoblasts were plated and cultured for 4 h under standard conditions and then exposed to bafilomycin (100 nm) for up to 24 h under standard conditions. B, Trophoblasts were plated and cultured for 4 h under standard conditions and then exposed to bafilomycin (100 nm) for up to 24 h under hypoxic conditions. Left panels of A and B show representative Western blots of cleaved PARP and Atg7. The data of 1 h exposure to bafilomycin were used for the quantification analyses. Second to the right panels of A and B show the summary graphs of densitometry of the Western blots. Data are presented as mean ±sd, with n = 3. *, P < 0.05 by Student's t test.

Fig. 6.

Fig. 6.

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The effect of autophagy inhibitor LY294002 on the apoptosis in trophoblasts. Trophoblasts were plated and cultured for 4 h and then exposed to hypoxia compared with standard conditions for 24 h with or without LY294002 (10 μm). Representative Western blot of cleaved PARP (cl-PARP) and LC3-II are shown on the left panel, and the summary graphs of the densitometry of the Western blots are shown in the middle and right panels. Data are presented as mean ±sd, with n = 3–6. *, P < 0.05 by two way ANOVA/Bonferroni's post hoc test.

Fig. 7.

Fig. 7.

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The effect of silencing Atg7 on the autophagy and apoptosis in trophoblasts. A, Expression of Atg7 in hypoxic trophoblasts compared with control. Trophoblasts were plated and cultured for 4 h under standard conditions and then exposed to hypoxia for 24 h. Left panel shows representative Western blot of Atg7. Right panel shows the summary graph of densitometry of Atg7 represented on the left panel. B and C, Trophoblasts were plated and cultured for 4 h under standard conditions and then transfected with two different types of synthesized double-stranded siRNA for Atg 7, siAtg7–1, and siAtg7–2 for 24 h under standard conditions. The cells were then transferred into hypoxic conditions for another 24 h, either with DMSO as control (B) or with bafilomycin (100 nm) (C). Left panels of B and C show the Western blots of Atg7, LC3-II, and cleaved PARP (cl-PARP). Second to the right panels of B and C show the summary graphs of densitometry of Atg7, LC3-II, and cl-PARP. The quantification was based on the results of siAtg7–2, and data are presented as mean ±sd, with n = 3. *, P < 0.05 Student's t test.

Western blotting

Primary human trophoblasts were lysed in radioimmunoprecipitation buffer (1% Nonidet P-40, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate in PBS) containing protease and phosphatase inhibitors (Sigma). Twenty micrograms of proteins per lane were separated by SDS-PAGE and transferred overnight at 150 mA at 4 C to polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA). The membrane was blocked in 5% nonfat dry milk in PBS with 0.05% Tween 20 for 1 h and then incubated overnight at 4 C or 4 h at room temperature, with one of the following primary antibodies: rabbit polyclonal anti-LC3-II (1:3000; Novus, Littleton, CO), rabbit monoclonal antiphospho-S6 ribosomal protein (1:2000; Cell Signaling Technology), rabbit monoclonal anti-cleaved poly(ADP-ribose) polymerase (PARP, 1:1000; Cell Signaling Technology), mouse monoclonal anti-p62 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit monoclonal anti-Atg7 (1:1000; Sigma), or goat polyclonal antiactin (1:3000; Santa Cruz Biotechnology) in 5% nonfat dry milk in PBS with 0.05% Tween 20. The blot was then incubated at room temperature for 1–2 h in horseradish peroxidase-conjugated donkey antimouse, donkey antirabbit, or donkey antigoat IgG secondary antibodies (1:10,000; Santa Cruz Biotechnology), as appropriate. The blots were washed and processed for chemiluminescence with SuperSignal West Dura extended duration substrate kit (Thermo Scientific, Rockford, IL). Densitometry of bands on films was assessed with Epichemi-3 software (UVP BioImaging Systems, Upland, CA) and normalized to actin levels, as noted in the figure legends.

Immunofluorescence

Primary human trophoblasts were fixed with 2% paraformaldehyde for 10 min and stained for p62 (1:200) and for nuclei with Draq 5 (5 μm; Biostatus, Shepsted, UK). Images were captured as previously described (22). The p62 signal was quantified by counting p62 puncta (diameter > 0.5 μm) in 10 randomly selected fields in each sample. The results are presented as the average number of puncta per nucleus.

Small interfering RNA (siRNA) silencing of Atg7 in primary human trophoblasts

Atg7 Silencer Select siRNA (no. s20650: GGAACACUGUAUAACACCAtt; s20651: CGCUUAACAUUGGAGUUCAtt) were purchased from Ambion (Norwalk, CT). Trophoblasts were transfected using DharmaFECT 1 (Dharmacon, Lafayette, CO) as described previously (33).

Statistical analysis

Data are presented as mean ± sd. All experiments were repeated at least three times, using trophoblasts isolated from three different placentas. Results are depicted by a representative experiment. One/two-way ANOVA with Bonferroni post hoc test or Student's t test was performed where appropriate, using KaleidaGraph software (Synergy Software, Reading, PA), version 4.1.0 for Macintosh and a P < 0.05 for significance.

Results

Autophagy occurs in cultured cytotrophoblasts

To compare levels of autophagy and apoptosis in primary cytotrophoblasts under standard conditions, we examined expression of the respective markers, LC3-II and cleaved PARP. After 20 h of culture, designated as 0 h, trophoblasts expressed detectable LC3-II (Fig. 1 B). After a change to fresh medium, the expression of LC3-II diminished 5-fold over the next 4 h but rose again by 8 h culture in the same medium. Interestingly, the expression level of cleaved PARP was inversely related to the level of LC3-II at each time point (Fig. 1 B), suggesting there was a balance between apoptosis and autophagy under standard conditions.

Hypoxia regulates autophagy in cultured cytotrophoblasts

The autophagy regulator mTOR is known to control nutrient transfer in trophoblasts (17, 34, 35), but whether mTOR functions in the autophagy pathway in cytotrophoblasts has not been assessed. To examine this possibility, we first exposed cytotrophoblasts to rapamycin, which inhibits mTOR kinase activity, and measured inactivation of the mTOR complex by assessment of downstream phosphorylation of ribosomal protein S6 (Fig. 1A). Although rapamycin inhibited accumulation of phosphorylated ribosomal protein S6, the drug had no effect on the steady-state expression level of LC3-II in primary cytotrophoblasts (Fig. 2A). This is likely due to the fact that induction of autophagy induces both LC3-II expression and degradation of LC3-II in the autolysosome (15). To uncouple these effects, we treated the cells with bafilomycin, a drug that prevents fusion of lysosomes and acidification of autophagosomes (Fig. 1A) to limit degradation of cargo, including LC3-II (15). Bafilomycin treatment yielded increased levels of LC3-II but had no effect on phosphorylation of ribosomal protein S6 (Fig. 2A). Moreover, coexposure of trophoblasts to rapamycin plus bafilomycin resulted in increased LC3-II levels, compared with exposure to bafilomycin alone (Fig. 2A).

Hypoxia induces apoptosis in human cytotrophoblasts in culture and in villous explants (3, 6, 22). To determine whether low oxygen tension also induces autophagy in these cells, we examined phosphorylation of ribosomal protein S6 after exposure to hypoxic conditions. Levels of phosphorylated ribosomal protein S6 decreased within 1 h and remained low for the 24 h exposure to hypoxia (Fig. 2B). Hypoxia also significantly decreased the levels of LC3-II in trophoblasts (Fig. 2B). We again used bafilomycin to determine whether the decreased expression of LC3-II occurred from inhibition of autophagosome generation or increased autophagic flux from increased degradation of LC3-II in the autophagosome (15, 36). We exposed trophoblasts cultured in standard conditions of 20% O2 or hypoxic conditions of less than 1% O2 to bafilomycin or DMSO vehicle control and assessed autophagic flux (Fig. 3, A and B). The LC3-II levels in bafilomycin were significantly higher in hypoxic compared with standard conditions (Fig. 3C), reflecting an increase in autophagic flux in hypoxia.

We next used immunofluorescence staining to assess another marker for autophagic flux, p62 (also known as SQSTM1/sequestosome 1), which interacts with LC3-II and is degraded during autophagy (Fig. 1A). We found p62 was present in two types of puncta in the cytoplasm including a high number of faint, approximately 0.1- to 0.2-μm-diameter bodies and brighter, approximately 0.5- to 1-μm-diameter bodies, as previously reported (37). We counted the approximately 0.5- to 1-μm p62 bodies and normalized these to the number of nuclei within each field and found fewer p62 puncta per nucleus in hypoxia compared with standard conditions (Fig. 4). Conversely, there were more p62 puncta per nucleus in the presence of bafilomycin in trophoblasts exposed to less than 1% oxygen than in cells cultured with bafilomycin in standard culture conditions (Fig. 4).

Bafilomycin blocks autophagic flux and induces apoptosis in cultured trophoblasts

Autophagy often has prosurvival effects, reducing the induction of apoptosis in homeostasis and in response to stress (38). We thus tested the hypothesis that blocking autophagy induces apoptotic cell death in trophoblasts. Indeed, in standard conditions bafilomycin blockage of autophagic flux induced apoptosis in trophoblasts, as shown by increased expression of cleaved PARP (Fig. 5A). We next sought to gain insights into mediators that provide the interregulation of autophagy and apoptosis. Atg7 has been reported to function in both of these processes. Atg7 is an ubiquitin-E1-like enzyme that plays an important role in elongation of preautophagosomal structures and has been reported to play a proapoptotic role during autophagic stress (39). We found that bafilomycin, which increased apoptosis as above, also enhanced expression of Atg7 in trophoblasts cultured in standard conditions (Fig. 5A). We next examined the effect of bafilomycin in trophoblasts under less than 1% oxygen conditions and found that bafilomycin increased the expression of cleaved PARP but not Atg7, compared with control (Fig. 5 B).

Phosphatidylinositol 3-kinase (PI3 kinase) inhibition blocks autophagic flux and induces apoptosis

We sought to confirm the results noted above by blocking autophagy at a different step in the pathway (Fig. 1A). Bafilomycin blocks a late stage of autophagy, but the PI3 kinase inhibitor LY294002 inhibits an early stage of autophagy by targeting preautophagosomal structures. Notably, LY294002 enhanced apoptosis in trophoblasts under both standard and hypoxic conditions (Fig. 6). Although LY294002 decreased LC3-II expression under standard conditions, it was unable to change the expression of LC3-II under hypoxia. In addition, the cleaved PARP levels in LY294002 were not different in less than 1% oxygen compared with 20% oxygen (Fig. 6), likely because LY294002 had already maximized apoptosis before exposure to hypoxia.

Silencing of Atg7 decreases autophagy and apoptosis in trophoblasts

Because hypoxia increased the levels of Atg7, compared with standard control (Fig. 7A), we next investigated the role of Atg7 in autophagy in trophoblasts under hypoxic conditions. Atg7 expression was significantly diminished by two different Atg7 siRNA but not by a control scrambled siRNA (Fig. 7, B and C). Knockdown of Atg7 did not detectably affect the expression of LC3-II or cleaved PARP in trophoblasts under low oxygen (Fig. 7B); however, in hypoxic trophoblasts exposed to bafilomycin, knockdown of Atg7 reduced LC3-II levels, reflecting less autophagy, and decreased cleaved PARP levels, reflecting reduced apoptosis (Fig. 7C).

Discussion

The data support the hypothesis that autophagy plays a prosurvival role in human trophoblasts. First, low oxygen tension reduced mTOR activity and increased autophagy in cytotrophoblasts. Second, inhibition of autophagy by bafilomycin resulted in even higher expression of two markers of autophagy, LC3-II and p62, in cytotrophoblasts exposed to hypoxia, indicating that hypoxia induces autophagic flux in villous trophoblasts. Third, bafilomycin also enhanced expression of Atg7, and the increase of this protein paralleled the increase in expression of cleaved PARP, a marker of apoptosis. Fourth, inhibition of PI3 kinase, which blocks formation of preautophagosomal structures, increased apoptosis under both hypoxia and standard conditions in the trophoblasts, compared with control. Finally, silencing of Atg7 reduced both apoptosis and autophagy, demonstrating that Atg7 is pivotal in trophoblast survival-demise determinations.

The mTOR pathway senses nutrient supply in multiple cells (11), including primary human trophoblasts (17). Notably, IGF-I and insulin increase amino acid transporter activity in trophoblast in an mTOR-dependent manner (17), and both amino acid transport and mTOR activity are reduced in pregnancies complicated by maternal protein deprivation (40) or fetal growth restriction (35, 41). The mTOR pathway is also a central regulator of autophagy (11). Although the placentas from uncomplicated term pregnancies show markers of autophagy (21, 42), this is the first study to dissect the role of the mTOR pathway during autophagy in human trophoblasts.

Although named for the protein's susceptibility to rapamycin, the mTOR can form two complexes, mTORC1 and mTORC2, of which only mTORC1 senses rapamycin. The mTORC1 complex regulates protein synthesis and growth in part by phosphorylation of downstream targets. We thus examined the expression of phosphorylated ribosomal protein S6 as a measure of rapamycin's inhibition of mTORC1 activity, and as predicted, we observed an mTOR-dependent signaling pathway. Importantly, the effect of hypoxia on human cytotrophoblasts is also to reduce phosphorylation of the S6 protein and, like rapamycin, to induce autophagy in cytotrophoblasts, albeit in a slower manner than rapamycin. Collectively our results indicate that hypoxia modulates the mTOR signaling pathway and enhances autophagy in primary cultures of human cytotrophoblasts.

Both apoptosis and autophagy are highly regulated, dynamic processes that maintain tissue homeostasis. Autophagy is recognized to play a prosurvival role in most cells (9), despite the previous assignment as the type II cell death pathway (43). Apoptosis as a true death pathway contributes to cell turnover and plays an important role in the tissue remodeling induced by stressors. Our data indicate that there is interplay between autophagy and apoptosis in stressed human cytotrophoblasts. Mediators that participate in both autophagy and apoptosis in other cells include B-cell CLL/lymphoma (Bcl)-2 and Bcl-XL, which bind to a BH3 domain to inhibit Beclin-1 (44–47). The Atg5 also plays a role in both processes because calpains cleave Atg5 to a fragment that regulates apoptosis (48). We herein show a dual role for Atg7 in the regulation of autophagy and apoptosis in hypoxic cytotrophoblasts. Silencing Atg7 reduces both autophagy and apoptosis in cytotrophoblasts, reflected by reduced LC3-II and cleaved PARP as measures of the two phenomena, respectively. Taken together, these observations support a cross talk of autophagy and apoptosis in cultured cytotrophoblasts.

Cytotrophoblasts and syncytiotrophoblasts display phenotypic variability in their responses to stressors. For example, syncytiotrophoblasts are more resistant to apoptosis than are cytotrophoblasts (1, 2). We have unveiled the presence, and begun to dissect the regulation, of autophagy in cytotrophoblasts and are showing there are interactions between the autophagic and apoptotic pathways that determine survival of cultured cytotrophoblasts. However, we acknowledge that culture conditions are unlikely to mimic the in vivo environment completely, and the role autophagy plays in the survival of cytotrophoblasts in situ is unknown. Moreover, the relationship between apoptosis and autophagy in syncytiotrophoblast might differ from that in cytotrophoblasts, whether observed in culture or in vivo. Nonetheless, we speculate that autophagy benefits placental function in vivo by eliminating cytotrophoblast organelles damaged by stressors, such as hypoxia. As an epithelial stem cell population, cytotrophoblasts are thereby spared cell death and are thereby available to fuse to form more syncytiotrophoblast, which in turn avoids receipt of damaged organelles like mitochondria that produce excess reactive oxygen species. Concurrently a pool of nutrients is available from the autophagic pathway, and these substrates could distribute to foster growth of villi and the fetus. These speculations offer testable hypotheses for future research.

Acknowledgments

This work was supported by National Institutes of Health Grant R01 HD-29190 (to D.M.N.) and The Foundation for Barnes-Jewish Hospital (to D.M.N.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
Atg
Autophagy-related protein
Bcl
B-cell CLL/lymphoma
DMSO
dimethylsulfoxide
LC3-II
Light chain 3-II
mTOR
mammalian target of rapamycin
mTORC
mTOR complex
PARP
poly(ADP-ribose) polymerase
PI3 kinase
phosphatidylinositol 3-kinase
siRNA
small interfering RNA.

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