Where Polarity Meets Fusion: Role of Par6 inTrophoblast Differentiation during PlacentalDevelopment and Preeclampsia
Tharini Sivasubramaniyam, Julia Garcia, Andrea Tagliaferro,Megan Melland-Smith, Sarah Chauvin, Martin Post, Tullia Todros,and Isabella Caniggia
Samuel Lunenfeld Research Institute (T.S., J.G., A.T., M.M.-S., S.C., I.C.), Mt Sinai Hospital, Toronto,Ontario, M5G 1X5, Canada; Departments of Obstetrics and Gynecology (I.C.), Physiology (T.S., M.M.-S.,S.C., M.P., I.C.), Faculty of Medicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada;Hospital for Sick Children (M.P.), Toronto, Ontario, M5G 1X8, Canada; and Department of Obstetricsand Gynecology (T.T.), Maternal-Fetal Medicine Unit, University of Turin, 10124 Italy
Trophoblast cell fusion is a prerequisite for proper human placental development. Herein weexamined the contribution of Par6 (Partitioning defective protein 6), a key regulator of cell po-larity, to trophoblast cell fusion in human placental development. During early placentation, Par6localized to nuclei of cytotrophoblast cells but with advancing gestation Par6 shifted its localizationto the cytoplasm and apical brush border of the syncytium. Exposure of primary isolated tropho-blasts to 3% O2 resulted in elevated Par6 expression, maintenance of tight junction marker ZO-1at cell boundaries, and decreased fusogenic syncytin 1 expression compared with cells cultured at20% O2. Treatment of choriocarcinoma BeWo cells with forskolin, a known inducer of fusion,increased syncytin 1 expression but decreased that of Par6 and ZO-1. Par6 overexpression in thepresence of forskolin maintained ZO-1 at cell boundaries while decreasing syncytin 1 levels. Incontrast, silencing of Par6 disrupted ZO-1 localization at cell boundaries and altered the expressionand distribution of acetylated �-tubulin. Par6 expression was elevated in preeclamptic placentasrelative to normotensive preterm controls and Par6 located to trophoblast cells expressing ZO-1.Together, our data indicate that Par6 negatively regulates trophoblast fusion via its roles on tightjunctions and cytoskeleton dynamics and provide novel insight into the contribution of this polaritymarker in altered trophoblast cell fusion typical of preeclampsia. (Endocrinology 154: 1296–1309,2013)
Cell-cell fusion, a process in which two cells merge theirplasma membrane and become a single cell, is a dy-
namic cellular event that is tightly regulated in a spatialand temporal manner (1–4). In humans, trophoblast cellfusion is a critical differentiation event essential for theestablishment of a functional placenta, a prerequisite forproper embryonic development and a successful preg-nancy. The syncytiotrophoblast (ST) layer is a unique ter-minally differentiated, multinucleated syncytium, charac-terized by the absence of mitotic bodies. It is unable toreplicate, and it is sustained by continuous fusion of un-
derlying cytotrophoblast (CT) cells, an event that allowsthe syncytium to remain functionally active (5).
Studies have identified a number of membrane andnonmembrane protein regulators of trophoblast cell fu-sion including syncytin 1, an envelope glycoprotein of ahuman endogenous retrovirus of the HERV-W family (6);transcription factor glial cell missing 1 (GCM1) that bindsto the syncytin 1 promoter (7); aspartate-specific cysteineprotease caspase 8 (8) and metalloprotease ADAM-12 (9).Additionally, whereas a variety of growth factors includ-ing epidermal growth factor (10) and vascular endothelial
has been shown to inhibit this cellular event (12). Further-more, physiological changes in oxygen, experienced by thedeveloping placenta, have been shown to regulate tropho-blast cell fusion whereby low O2 exerts an inhibitory effect(13–16), thereby maintaining trophoblast cells in an un-differentiated proliferative state.
Preeclampsia, a serious disease affecting 5%–8% ofpregnancies, remains one of the major contributors of ma-ternal and perinatal morbidity and mortality (17). Studiesconducted in vivo and in vitro have demonstrated thathypoxia and oxidative stress typifies the preeclamptic (PE)placenta (18–22). Altered trophoblast cell fusion found inpreeclampsia contributes to the aberrant cell turnover typ-ical of this pathology and is attributed, in part, to theoxidative stress status experienced by the placenta (5). Inaddition, studies have shown down-regulation of the tran-scription factor GCM1 and its associated target gene, fu-sogenic protein syncytin 1, in PE placentas (23, 24).
Within the human placenta, tight junctions betweenpolarized progenitor CT cells maintain these cells in anundifferentiated state; however, in order for these cells todifferentiate and fuse to form the syncytium, such junc-tions need to be disrupted, leading to loss of polarity. Cellpolarity is an intrinsic property of all eukaryotic cells thathas been implicated in a variety of cellular processes in-cluding differentiation, proliferation, and morphogenesis(25, 26). Epithelial cells possess apical-basolateral polar-ity that results from the asymmetric distribution of lipidsand proteins to either their apical or basolateral plasmamembrane surfaces (27, 28). Molecules involved in cyto-skeleton reorganization and subcellular organelles such asthe trans-Golgi complex that functions as a major sortingsite for newly synthesized plasma-membrane proteins, arealso important in regulating epithelial cell polarity (29–32). Notably, regulators of cell polarity are necessary forthe establishment and maintenance of tight junctions. Inparticular, partitioning defective protein 6 (Par6), plays arole in the creation of apical-basolateral polarity throughthe formation and maintenance of tight junctions (29–32). Par6 exists in three isoforms in mammals: A/C, B, andD/G. Notably, Par6A/C isoform, unlike its cognates, hasbeen reported to play a role in the loss of tight junctionsduring cancer progression via a mechanism involving theTGF�-signaling pathway (33, 34).
Although it is established that several factors, includingoxygen, guide trophoblast cell differentiation, the contri-bution of cell polarity regulators such as Par6 to tropho-blast cell fusion remains elusive. Herein, we report thatPar6 plays a role in regulating trophoblast cell fusion in thehuman placenta. In particular, we demonstrate that Par6expression is guided by oxygen and acts as a negative reg-
ulator of trophoblast fusion by maintaining tight junctionintegrity and cytoskeletal rearrangements. Additionally,we show that increased Par6 expression in PE placentasmay contribute, in part, to the aberrant trophoblast fusiontypical of this pathology.
Materials and Methods
Placental tissue collectionInformed consent was obtained from each individual, and
tissue collection was carried out in accordance with participatinginstitutions ethics’ guidelines (Ethics guidelines of the Universityof Toronto’s Faculty of Medicine and Mount Sinai Hospital) byBiobank of Mount Sinai Hospital and by the O.I.R.M-Sant’AnnaHospital, University of Turin, Italy. Human placental tissuesranging from 6-40 weeks of gestation (n � 25) were obtainedfrom elective pregnancy terminations by dilatation and curet-tage. PE placentas (n � 25) were selected based on the AmericanCollege of Obstetricians and Gynecologists clinical and patho-logical criteria (17). Calcified, necrotic, and visually ischemicareas of the placental tissue were omitted from sampling. Pla-cental tissues obtained from age-matched preterm deliveries in-cluded as controls (PTC, n � 18) did not show signs of pre-eclampsia or other placental pathology. PE patients delivered atan average gestational age of 30 weeks and exhibited high bloodpressure averaging 175/109 (systolic/diastolic) with the presenceof proteinuria (average 3.1 g/d). The term control and PTC pa-tients delivered at an average gestational age of 38 and 31 weeks,respectively, had normal blood pressure values (122/74 and 118/69, respectively) with no proteinurea. Immediately after deliv-ery, tissues were either snap frozen in liquid nitrogen or fixed inparaformaldehyde and embedded in paraffin and processed forhistochemical analysis.
Primary trophoblast cell isolationPrimary trophoblast cell isolation was performed as previ-
ously described (35). Placentas (n � 14) were obtained afterelective cesarean sections from uncomplicated term pregnanciesin accordance with the established guidelines of the ethical com-mittee of Mount Sinai Hospital and the University of Toronto’sFaculty of Medicine. Briefly, placental tissue was digested with0.05 mM trypsin (GIBCO 27250-018; Invitrogen, Carlsbad,California) and 0.008 mM DNase I (SIGMA DN25; Sigma-Al-drich Corp. St. Louis, Missouri) at 37°C in Hank’s buffered saltsolution. The obtained cell suspension was subsequently layeredon top of a discontinuous 5%–70% Percoll (Sigma) gradient andafter a centrifugation step, the intermediate layer containing CTswas removed and washed with DMEM (Invitrogen). Cells werecultured at a density of 2.5 � 106 cells/well in six well-plates incomplete DMEM containing 10% heat-inactivated fetal bovineserum (Sigma) and 10,000 U/ml of penicillin and streptomycin at37°C and maintained at standard conditions (20% oxygen, 5%CO2 in 95% air).
Cell viability was determined by trypan blue exclusion (In-vitrogen). Cell purity was assessed by immunofluorescence (IF)staining for cytokeratin, an epithelial cell lineage marker andvimentin, a mesenchymal cell lineage marker. After 24 hours,cells were maintained in either standard conditions or in an at-
Endocrinology, March 2013, 154(3):1296–1309 endo.endojournals.org 1297
mosphere of 3% O2/92% N2/5% CO2 for 12, 24, 48, and72 hours at 37°C. Cells were either collected at each time pointin radioimmune precipitation assay buffer for Western blot (WB)analysis or fixed in methanol to perform immunostaining.
Choriocarcinoma cell culturesHuman choriocarcinoma BeWo cells (American Type Cul-
ture Collection (ATCC), Manassas, Virginia) were seeded at adensity of 4 � 105 cells/well on six-well plates and cultured incomplete Ham’s F12K medium (ATCC) supplemented with 2mM L-glutamine and 10% heat-inactivated fetal bovine serumwith penicillin and streptomycin at 37°C. After overnight incu-bation, 25 �M of forskolin (F) (Sigma-Aldrich Corp.), a knowninducer of trophoblast fusion, was added to the cells. Controlvehicle-treated cells were incubated with culture medium con-taining 0.1% dimethyl sulfoxide (DMSO). Cells were collectedat 12, 24, 48, and 72 hours for either Western blot analysis orfixed in 3.7% formaldehyde for immunofluorescent analysis.
Loss- and gain-of-function studiesBeWo cells were plated at a density of 2.0 � 105 in six-well
plates and then cultured for 24 hours in complete media at 37°Cunder standard conditions (5% CO2 in 95% air). For overex-pression experiments, BeWo cells were transfected with 1.5 �g ofempty pcDNA3.1 or Par6a-encoding pcDNA3.1 plasmid. ThecDNA encoding the open reading frame of Par6A (Open Bio-systems, Huntsville, Alabama) was directionally cloned into apcDNA3.1/Hygro (�) vector (Invitrogen). Forward and re-verse primers used encoded a HindIII and a BamHI restrictionsite, respectively. The primers were as follows: forward, 5�-CCCAAGCTTGCCCGGCCGCAGAGGACTC-3�; reverse, 5�-CGGGATCCTCAGA GGCTGA AGCCACTACC-3�. For si-lencing experiments, cells were transfected with 60 nM of twodifferent Silencer Select small interfering RNA (siRNA) directedagainst the isoform A of human Par6 gene, or control scramblesequence (SS) (Ambion Inc, Austin, Texas) for 48 hours. Trans-fections were performed using Lipofectamine transfection re-agent (Invitrogen) based on the manufacturer’s protocol in theabsence of antibiotics. Twenty-four hours after Par6 overexpres-sion, cells were treated with either 25 �M forskolin or 0.1%DMSO for an additional 48 hours. Cells were either collected forWestern blotting or fixed in 3.7% formaldehyde for immuno-fluorescent analysis. Conditioned media and cell lysate werealso collected for �-human chorionic gonadotropin (hCG)analysis using a hCG Human ELISA kit (catalog no.ab100533; Abcam; Cambridge, Massachusetts) according tothe manufacturer’s instructions.
tion; WB, 1:500], rabbit polyclonal anti-ZO-1 (H300) [IF,1:100], and goat polyclonal anti-�-actin (I-19) [WB, 1:1000]antibodies were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, California). Rabbit polyclonal andante-acetylated�-tubulin (LYS40) [IF, 1:100], mouse monoclonal anti-E-cad-herin (HECD-1) [WB, 1:1000; IF, 1:400] and rabbit antidesmo-plakin [IF, 1:100] antibodies were obtained from Abcam. Rabbitpolyclonal GCM1 antibody [WB, 1:3000; IF, 1:1000] was ob-tained from Aviva Systems Biology (San Diego, California) andmouse monclonal hCG antibody [IF, 1:100] was purchased from
Abcam. Rabbit monoclonal antibodies generated against a pep-tide mapping within an integral region of syncytin of humanorigin were raised in our laboratory [IF, 1:100; WB, 1:400] (36).Secondary antibodies were horseradish peroxidase-conjugateddonkey antigoat, goat antirabbit, and goat antimouse IgG (SantaCruz Biotechnology) or biotinylated antirabbit IgG (Vector Lab-oratories, Burlingame, California); Alexa Fluor 594 conjugateddonkey antigoat/antirabbit/antimouse and Alexa Fluor 488-conjugated donkey antigoat/antirabbit [IF, 1:200] were fromInvitrogen.
Western blot analysisWestern blotting was performed as previously described (37).
Membrane blots were visualized by enhanced chemilumines-cence (PerkinElmer Inc., Waltham, Massachusetts). All Westernblots were confirmed for equal protein loading by probing for�-actin. For quantification purposes, bands of interest were an-alyzed using CanoScanLiDE20 image scanner (Canon Canada,Inc, Mississauga, Ontario, Canada).
ImmunofluorescenceImmunofluorescence staining was performed as previously
described (38). In brief, sections of placental tissue were depar-affinized by xylene, followed by hydration through a decreasinggraded concentration of ethanol/water. Antigen retrieval wasperformed using 10 mM sodium citrate buffer solution. Toquench endogenous fluorescence, slides of placental tissue wereplaced in 0.3% Sudan black in 70% ethanol for 30 min at roomtemperature. For BeWo and primary isolated trophoblast cells,cells were permeabilized with 0.2% Triton X-100 (Bioshop Can-ada, Inc., Burlington, Ontario, Canada) in PBS. Both placentalsections and cells were preincubated with 5% normal horse se-rum diluted in PBS to block nonspecific binding. Placental sec-tions/cells were then incubated overnight at 4°C with primaryantibody. For negative controls, the primary antibodies werereplaced by a corresponding concentration of goat, mouse, orrabbit IgG. The following day, sections were washed in PBS andincubated with fluorescence-conjugated Alexa Fluor secondaryantibodies (1:200) for 1 hour at room temperature. The sections/cells were then counterstained with DAPI (4�,6-diamino-2-phe-nylindole) to detect the nuclei. Fluorescence images were cap-tured using the DeltaVision Deconvolution microscopy withz-stacking (Applied Precision, LLC, Issaquah, Washington). Fif-teen immunofluorescence images of forskolin-treated BeWo cellsoverexpressing Par6 or the control empty vector were capturedusing the deconvolution microscope. The images were thenmounted on a grid. Nine grid squares were randomly selected ineach image. The total number of nuclei in the selected boxes andthe number of lateral borders observed by the presence of ZO-1expression were manually counted. The analysis was performedon the proportion of ZO-1 at the lateral borders relative to thenumber of nuclei. Four different individuals were given the taskof scoring the images, and an average of the proportion of ZO-1per nuclei for each group was calculated. PE and PTC placentalsections were stained with hematoxylin and eosin (H&E) andimaged by light microscopy (Leica Microsystems, Inc).
Statistical analysisQuantification of Western blots was accomplished by densi-
tometry using Image Quant 5.0 software (Molecular Dynamics,
1298 Sivasubramaniyam et al Par6 and Trophoblast Cell Fusion Endocrinology, March 2013, 154(3):1296–1309
Piscataway, New Jersey). Expressions of all proteins of interestwere normalized to the housekeeping gene, �-actin. Statisticaltests were carried out using GraphPad Prism 4 software (SanDiego, California). For comparison of data between two groups,Mann-Whitney, Wilcoxon signed rank test, and paired or un-paired t test were performed, where applicable. For comparisonamong multiple groups, one-way ANOVA with post hoc Dun-nett’s or Newman-Keuls test was performed, where applicable.Statistical significance was defined as P � .05, and all data arerepresented as mean � SEM.
Results
Par6 expression in first-trimester human placentasWe first examined Par6 spatial and temporal expres-
sion during early placental development. Immunoblotanalysis of human placental lysates across gestations re-vealed a significant increase in Par6 protein levels at 10-15weeks when compared with 6-9 weeks of gestation andterm (Figure 1A). Immunofluorescence (IF) analysis dem-onstrated that in early first trimester (5 wk) Par6 localized
primarily to CT cells, displaying a prevalent nuclear lo-calization (Figure 1B). As gestation progresses (8 wk) Par6localization shifted to the cytoplasm of both CT and STlayers and at 12 weeks of gestation, Par6 was predominantlyfound at the apical brush border of the ST layer, which wasidentified by staining with placental alkaline phosphatase, atypical apical brush border marker (data not shown). CTcells were identified by staining with E-cadherin (Figure 1B).Ofnote,Par6-positive immunoreactivitywasalsodetected ina small subset of cells making up the villous mesenchyme(Figure 1B).
Low oxygen increases Par6 expression in isolatedtrophoblast cells
Because early placentation is associated with physio-logical changes of oxygen, we next examined the effect ofO2 on Par6 expression using isolated term trophoblastcells. Purity of isolated trophoblast cells was establishedby immunofluorescence analysis for cytokeratin 7, amarker of epithelial cells, and vimentin, a marker of mes-
Figure 1. Expression of Par6 during Early Placental Development. A, Representative Par6 immunoblot from placental tissue throughout gestation(6-9 wk of gestation, n � 6) (10-15 wk of gestation, n � 15) and term placentas (37-40 wk of gestation, n � 4). Densitometric analysis of Par6protein expression normalized to �-actin. * P � .05, unpaired t test. B, Representative images showing dual localization of Par6 (green) and E-cadherin (red) in sections of first trimester placental tissues. Nuclei were visualized with DAPI (blue). Magnification, �20 and �100. Arrow depictsnuclear localization of Par6.
Endocrinology, March 2013, 154(3):1296–1309 endo.endojournals.org 1299
enchymal cells. Most isolated cells (98%) were positive forcytokeratin 7 and negative for vimentin (data not shown).
Previous studies have established that primary tropho-blast cells undergo spontaneous fusion in culture as they ac-quire the expression of syncytin 1, an established fusogenicmarker (3, 39), and that low O2 prevented this fusion,thereby maintaining the cells in an undifferentiated state(14). In line with previous reports, exposure of trophoblastcells to 20% O2 resulted in a significant increase in syncytin1 expression as a function of time (Figure 2A). No significanttemporal change in syncytin 1 expression was noted whencells were maintained at 3% O2. In contrast, exposure ofprimary isolated cells to 3% O2 increased Par6 expression ina time-dependent fashion (Figure 2B), whereas Par6 expres-sion was constant in cells cultured at 20% O2. IF corrobo-
rated these immunoblot findings (Supplemental Figure 1Apublished on The Endocrine Society’s Journals Online website at http://endo.endojournals.org). At 3% O2, Par6 local-ized to the cytoplasm and cell boundaries of trophoblast cellswhereas syncytin 1 immunoreactivity was absent. Hence, inconditions of low oxygen, Par6 expression inversely corre-lates to that of syncytin 1.
Because the biogenesis of tight junctions is central to theestablishment and maintenance of apical-basolateral po-larity (31, 40), we next examined the expression of ZO-1,a tight junction marker, in primary isolated trophoblastcells kept at different O2 tensions for 12 or 72 hours. IFshowed that after 12 hours of culture, ZO-1 localized tocell boundaries in cells kept at either 3% O2 and 20% O2
(Supplemental Figure 1B, left panels). After 72 hours of
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Figure 2. Effect of Oxygen on Par6 and Syncytin 1 Expression in Primary Isolated Trophoblast Cells. A, Representative immunoblot for syncytin 1in cells maintained at 20% and 3% O2 for 0, 24, 48, and 72 hours. Densitometric analysis of syncytin 1 expression normalized to �-actin andexpressed as a fold change relative to T � 0 hours (C). *** P � .0001 and * P � .05, one-way ANOVA with post hoc Dunnett’s multiplecomparison test; and ** P � .0022, Mann-Whitney test, n � 7 independent experiments. B, Representative immunoblot of Par6 expression in cellsmaintained at 20% O2 and 3% O2 for 0, 24, 48, and 72 hours. Densitometric analysis of Par6 expression normalized to �-actin and expressed as afold change relative to T � 0 hours. ** P � .0012 and * P � .05, One-way ANOVA with post hoc Dunnett’s multiple comparison test; and ***P � .0087 and **** P � .0317, Mann-Whitney test, n � 7 independent experiments. C, Representative immunoblot of ZO-1 and E-cadherinexpression in cells maintained at either 20% or 3% O2 for 0 and 48 hours.
1300 Sivasubramaniyam et al Par6 and Trophoblast Cell Fusion Endocrinology, March 2013, 154(3):1296–1309
culture at 3% O2, Par6 and ZO-1 predominantly localizedto the cell boundaries (Supplemental Figure 1B, bottomright panel), whereas at 20% O2, Par6 immunoreactivitywas reduced and had a cytoplasmic localization, andZO-1 was absent from cell boundaries (Supplemental Fig-ure 1B, upper right panel). Western blotting revealed thatZO-1 and E-cadherin protein levels decreased at 20% O2
in a time-dependent fashion, whereas their expression wasmaintained at 3% O2 (Figure 2C), corroborating the IFfindings.
Par6 functions as a negative regulator oftrophoblast cell fusion
Choriocarcinoma BeWo cells were used as an in vitromodel to establish the direct involvement of Par6 in tro-
phoblast cell fusion. BeWo cells typically require treat-ment with forskolin to undergo fusion (7). Hence, we ex-amined Par6 expression in BeWo cells after forskolintreatment. Similar to primary isolated trophophoblastcells, immunoblotting revealed a time-dependent increasein syncytin 1 protein expression after exposure to forsko-lin (25 �M forskolin � F) when compared with controlcells treated with vehicle (0.1% DMSO) (Figure 3A, leftpanel). Forskolin-induced syncytin 1 expression inverselycorrelated to that of Par6, indicating a potential role forPar6 in trophoblast fusion (Figure 3A, right panel). Im-munofluorescence analysis demonstrated that in vehicle-treated BeWo cells, Par6 localized to the nucleus, cyto-plasm and sparsely at cell boundaries, whereas ZO-1 was
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Figure 3. The Effect of Forskolin (F) on Par6, Syncytin 1, and ZO-1 expression in BeWo Choriocarcinoma Cells. A, Left panel shows arepresentative immunoblot of syncytin 1 protein expression in BeWo cells after treatment with 25 �M forskolin (F) or 0.1% DMSO vehicle-control(V) for 12, 24, 48, and 72 hours. Densitometric analysis of syncytin 1 protein expression was normalized to �-actin and expressed as a fold changerelative to vehicle-treated cells. * P � .05 and *** P � .00051, Mann Whitney t test, n � 6 independent experiments carried out in duplicate.Right panel shows a representative immunoblot of Par6 protein levels after treatment of BeWo cells with 25 �M F or 0.1% DMSO at the timepoints indicated above. Densitometric analysis of Par6 protein levels was normalized to �-actin and expressed as a fold change relative to vehicle-treated cells. * P � .05, Mann Whitney t test, n � 6 independent experiments carried out in duplicate. B, Left panel shows a representativeimmunoblot of Par6, E-cadherin, and GCM-1 protein expression in cells transfected with either Par6 siRNA (S1 and S2) or a control SS. Right panel,densitometric analysis of Par6 expression normalized to �-actin and expressed as a fold change relative to SS control. ** P � .01 and * P � .05,one-way ANOVA with post hoc Newman-Keuls multiple comparison test, n � 4 independent experiments carried out in triplicate.
Endocrinology, March 2013, 154(3):1296–1309 endo.endojournals.org 1301
present at the cell borders (Figure 4A, upper panels). Afterforskolin treatment, Par6 immunoreactivity markedly de-creased, which was accompanied by a loss of ZO-1 fromthe cell boundaries (Figure 4A, lower panels).
To establish a direct involvement of Par6 in trophoblastcell fusion, we next conducted a series of loss- and gain-of-function studies. Par6 knockdown using two differentPar6 siRNA Duplexes (S1 and S2) resulted in a Par6 pro-tein reduction of 30%–40% (Figure 3B). This partialknockdown of Par6 was sufficient to loose ZO-1 immu-noreactivity from cell boundaries (Figure 4B). This wasaccompanied by a decrease in E-cadherin expression, anestablished marker of adherens cell junctions (41, 42) (Fig-ure 3B). Interestingly, Western blot and immunofluores-cence analyses showed that partial silencing of Par6 re-sulted in increased GCM1 (glial cells missing homolog 1)expression, the transcriptional factor that regulates syn-cytin 1 promoter activity (Figures 3B and 4C) (43). Thiswas accompanied by increased positive immunoreactivityfor syncytin 1 (Figure 4C, lower panels). In addition, par-tial silencing of Par6 was also associated with disappear-ance of desmoplakin from intracellular boundaries (Fig-ure 4D, upper panel) as well as increased expression of�-hCG, a known product of syncytialization(Figure 4D,lower panel). We then examined the consequence of Par6overexpression on syncytin 1 expression, ZO-1 localiza-tion, and secretion of �-hCG. Immunoblotting showedthat forskolin-treated BeWo cells transfected with a Par6pcDNA expression construct (Par6) had, indeed, in-creased amounts of Par6 (Figure 5A, left panel) when com-pared with cells transfected with empty vector (EV). Par6overexpression reduced forskolin-induced syncytin 1 ex-pression and restored E-cadherin expression, which wasreduced by forskolin (Figure 5A, right panel). Moreover,Par6 overexpression also decreased the forskolin-inducedsecretion of �-hCG in both conditioned media (Figure 5B,left panel) and cell lysates (Figure 5B, right panel). Also,Par6 overexpression maintained ZO-1 at the cell bound-aries (Supplemental Figure 2A, right panel). Semiquanti-tative analysis revealed that a higher proportion of tightjunctions were maintained at cell boundaries after fors-kolin treatment in cells overexpressing Par6 comparedwith empty vector (Par6: F, 0.32 � 0.02 vs EV; F, 0.19 �0.01, P � .0001). As an internal control, we overexpressedPar6 construct tagged to the FLAG epitope to assess thespatial localization of Par6 in BeWo cells. Immunohisto-chemical analysis revealed Par6 localization to both thecytoplasm as well as to cell boundaries (Supplemental Fig-ure 2A, left panel).
Previous studies have indicated an intimate link be-tween cell polarity and the cytoskeleton, ie, loss of cellboundaries in epithelial cells resulted in loss of cell polarity
and changes in cytoskeletal organization and composition(44, 45). Hence, we performed colocalization studies ofPar6 and acetylated �-tubulin, a marker of stabilized mi-crotubules, in forskolin-treated BeWo cells. In line withour aforementioned findings, IF revealed a decreased ex-pression of Par6 after forskolin treatment that was asso-ciated with an increased expression and change in the dis-tribution of acetylated �-tubulin, ie, elongation to the cellperiphery (Supplemental Figure 2B). Next, we assessed theexpression of acetylated �-tubulin in cells after siRNAsilencing of Par6 in BeWo cells. Par6 knockdown (eitherwith S1 or S2 siRNA) decreased Par6 expression relativeto scrambled siRNA (SS), and this reduction in Par6 trig-gered a cellular redistribution of acetylated �-tubulin sim-ilar to that found in forskolin-treated cells (SupplementalFigure 2C).
Par6 expression in PE placentasIt has been previously reported that placentas from PE
pregnancies are characterized by altered trophoblast cellfusion (5, 23, 24). Therefore, we examined the expressionof Par6 in PE placentas. Placentas from healthy age-matched preterm (PTC) deliveries were used as controls.Immunoblot analysis revealed a significant increase inPar6 protein expression in PE placentas when comparedwith PTC placentas (Figure 6). IF demonstrated a strongpositive signal for Par6 within the trophoblast layer of PEvilli; Par6 localized within the nucleus and cytoplasm oftrophoblast cells as well as sparsely to the apical brushborder of the ST (Supplemental Figure 3, left panels). In-terestingly, Par6 expression in PE placentas was associ-ated with increased maintenance of ZO-1 around tropho-blast cells (Supplemental Figure 3, left panels). Hardly anypositive signal for Par6 and ZO-1 was noted in placentalsections from PTC (Supplemental Figure 3, left panels).We additionally performed H&E analysis to assess themorphology of the placental structure in normal, PTC,and PE placentas. H&E sections of PE presented in-creased vasculature and increased blebbing of villi com-parent with PTC (Supplemental Figure 3).
Discussion
Whereas most studies have focused on the contribution ofPar6 to the grand scheme of cell polarity, its impact on celldifferentiation in developing human organs remains to beestablished. Herein we report, for the first time, that Par6is temporally and spatially expressed during early humanplacental development and that it negatively regulates tro-phoblast fusion in an oxygen-dependent manner by main-taining tight junction integrity and controlling cytoskel-
1302 Sivasubramaniyam et al Par6 and Trophoblast Cell Fusion Endocrinology, March 2013, 154(3):1296–1309
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Figure 4. The Effect Of Silencing Par6 on ZO-1, GCM1, and Syncytin 1 Localization in BeWo Choriocarcinoma Cells. A, BeWo cells treated withvehicle DMSO (V, vehicle) and forskolin (F) for 48 hours were stained for Par6 (red) and ZO-1 (green). Nuclei are counterstained with DAPI (blue).Magnification, �100. (n � 3 independent experiments carried out in triplicate). B, Representative images of cells after transfection with control SSor with Par6 siRNA (S1) for 48 hours and stained for Par6 (red) and ZO-1 (green). C, Representative images of cells after transfection with controlSS or with Par6 siRNA (S1) for 48 hours and stained for GCM1 (green, top panel) and syncytin 1 (red, bottom panel). D, Representative images ofcells after transfection with control SS or with Par6 siRNA (S1) for 48 hours as well as after forskolin treatment (48 h) followed by staining fordesmoplakin (green, top panels) and �-hCG (green, bottom panels). Nuclei are counterstained with DAPI (blue). Arrows indicate cell boundary.Magnification, �100 (n � 3 independent experiments carried out in triplicate).
Endocrinology, March 2013, 154(3):1296–1309 endo.endojournals.org 1303
etal dynamics. Moreover, we demonstrate that Par6expression is up-regulated in preeclampsia, thereby con-tributing to the aberrant trophoblast fusion found in thispathology.
Par6 expression has been reported in the human termplacenta (46), but no studies have systematically examinedthe expression and function of Par6 during the develop-ment of this organ and in pregnancy-related disorders.Our in vivo findings demonstrate that Par6 protein levelsand spatial distribution change as a result of advancinggestation, implicating a dynamic role for this polarity
marker in guiding trophoblast differentiation. Our obser-vation of a predominant nuclear localization of Par6 inproliferating CT cells during early first trimester agreeswith previous reports showing Par6 localization in thenucleus of MDCK and HeLa cells (29, 47). The nuclearlocalization of Par6 may imply a regulatory role for thispolarity marker in CT proliferation. Such a regulatory rolefor Par6 in proliferation has been suggested for mammaryepithelial cells (48). Further experiments are required tounderstand the role of Par6 in the nucleus of early humantrophoblast cells. We also observed that Par6 localized to
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Figure 5. The Effect of Forskolin (F) Treatment on Syncytin 1 and �-hCG Expression after Par6 Overexpression in BeWo Choriocarcinoma Cells. A,Top left panel shows a representative immunoblot of Par6 protein expression in cells treated with forskolin (F) and control DMSO (V, vehicle) for48 hours after a 24-hour transfection with either pcDNA3.1 empty vector (EV) or pcDNA3.1Par6 (PAR6) construct. Bottom left panel,Densitometric analysis of Par6 expression was normalized to �-actin and expressed as a fold change relative to control EV. * P � .0313, Wilcoxonsigned rank test, n � 5 independent experiments carried out in duplicate. B, Top right panel shows a representative immunoblot of syncytin 1 andE-cadherin protein levels in cells treated with F and DMSO (V, vehicle) for 48 hours after a 24-hour transfection with EV or Par6 construct. Bottomright panel, Densitometric analysis of Synctin 1 expression was normalized to �-actin and expressed as a fold change relative to control EV. * P �.0313, Wilcoxon signed rank test and ** P � .0476, Mann-Whitney test, n � 5 independent experiments carried out in duplicate. B, �-hCGreleased in the conditioned media (left panel) and cell lysates (right panel) as measured by ELISA after treatment of cells with F and control DMSO(V, vehicle) for 48 hours after a 24-hour transfection with either pcDNA3.1 (EV) or pcDNA3.1Par6 (PAR6) construct. * P � .05, Mann-Whitney test,n � 3 independent experiments carried out in duplicate.
1304 Sivasubramaniyam et al Par6 and Trophoblast Cell Fusion Endocrinology, March 2013, 154(3):1296–1309
the apical brush border of the syncytium toward the endof first trimester. The apical brush border of the syncytiumis involved in the transepithelial transfer of metabolitesfrom the maternal to the fetal circulation as a result of thepolarized distribution of transporters expressed on theapical and basal membranes of the ST layer (49, 50). Be-cause Par6 has been implicated in apical-basal polarity ina variety of systems (25, 31, 51), its localization at theapical site of the syncytium may contribute to the asym-metric distribution of these transporters. Alternatively,Par6 at the apical brush border may represent remnants ofcytosolic Par6 when CT cells fuse into the overlying STduring syncytial growth (52). Of note, we observed ex-pression of Par6 in the mesenchyme of chorionic villi. Al-though no major changes in Par6 staining in this compart-ment were observed throughout gestation, this maycontribute to the increased Par6 expression in placentallysates. The fusion of CT cells into the overlying syncytiallayer is not limited to fusogenic proteins that merge theopposing membranes, but also includes remodeling ofmolecules that link adjacent cells. Indeed, previous studieshave reported down-regulation of E-cadherin and desmo-plakin during syncytialization (41, 53). Studies usingMDCK cells, mammary gland epithelial cells, and epithe-lial cells from Caenorhabditis elegans have shown a rolefor Par6 as a regulator of tight junction integrity (33, 40,57). Our loss- and gain-of-function studies in BeWo cells,an established model suitable to study trophoblast syncy-tialization (54–56), show that Par6 is a negative regulatorof trophoblast cell fusion by preserving tight junction in-tegrity and cytoskeletal dynamics. Partial silencing of Par6
led to a loss of tight junctions from the cell boundaries.This is in line with previous studies using Drosophilia, C.elegans, and mammalian cells in which deletion/mutationof Par6 resulted in loss and/or fragmentation of apicaljunctions as well as failure in the localization of apicaljunction proteins, leading to loss of apical-basal polarity(31, 40, 57). Notably, partial silencing of Par6 resulted inincreased cytoplasmic GCM1, Syncytin1, and �-hCG,which were associated with a redistribution of ZO-1 fromcell boundaries to nuclei. Studies have postulated that theswitch of ZO-1 from tight junctions to the nucleus is in-versely related to the extent and maturity of cell contacts,and ZO-1 nuclear localization occurs before and duringthe maturation and remodeling of cell-cell contacts (58).Because polarized CT cells need to lose these lateral bor-ders to undergo fusion, it is tempting to speculate that thedecrease in Par6 during trophoblast fusion directly pro-motes the loss of tight junction integrity in order for fusionto progress. Our finding that partial Par6 silencing inBeWo cells decreased the expression of desmoplakin andE-cadherin, a cell adhesion molecule, supports this ideaand is in agreement with a recent report showing that theformation of a multinucleated syncytium is associatedwith a decrease in E-cadherin expression (42). Addition-ally, our data demonstrate that inhibition of Par6 inducesa remodeling of the microtubular network as indirectlyvisualized by acetylated �-tubulin staining. Indeed, reor-ganization of the cytoskeleton is a general cellular phe-nomenon of cell-cell fusion in a variety of systems (8, 59).For example, acetylated �-tubulin has been found to beinvolved in promoting myoblast fusion and HIV type Ienvelope-dependent cell fusion (60, 61). Our study is thefirst to show a link between acetylated �-tubulin and tro-phoblast syncytialization. This finding agrees with reor-ganization of microtubular network in cell fusion being auniversal trait. Other studies have demonstrated that micro-tubulesare implicated incell shapeandmembranecurvature,events thatare likelyrequiredforfusionofadjacentcellmem-branes (8, 62, 63). Our finding of maintenance of ZO-1 atcell boundaries and decreased syncytin 1 expression afterforskolin treatment in Par6-overexpressing BeWo cells fur-ther supports a function for Par6 as a negative regulator oftrophoblast fusion. This is consistent with the decreased se-cretion of �-hCG, a marker of syncytial differentiation.Taken together, our data demonstrate a direct regulatoryeffect of Par6 on ZO-1 spatial distribution and changesin cytoskeleton reorganization, leading to inhibition ofsyncytialization.
Our present studies with primary isolated trophoblastsindicate that under hypoxic conditions, a condition re-ported to inhibit trophoblast fusion and maintaining cellsin an undifferentiated state, Par6 levels are elevated and
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Figure 6. Expression of Par6 in PE and PTC Placentas. Representativeimmunoblot of Par6 expression in PE and age-matched PTC placentas.Whole-cell lysate of Caki-1 renal adenocarcinoma cells was used as apositive control (�). Densitometric analysis of Par6 expression wasnormalized to �-actin in PE placentas (n � 16) and age-matched PTC(n � 10) placentas. ** P � .0041, Mann Whitney test.
Endocrinology, March 2013, 154(3):1296–1309 endo.endojournals.org 1305
both Par6 and ZO-1 localize to cell-cell boundaries. Thisis the first study to demonstrate that the expression of Par6is oxygen dependent. Under standard (20% O2) condi-tions and after forskolin treatment, which promotes fu-sion, Par6 and ZO-1 are lost from the cell boundaries, amechanism previously alluded to as lateral cell borders arelost due to syncytialization. In line with our findings, it hasbeen demonstrated that before syncytialization ZO-1 lo-calized initially to intercellular boundaries (4). The pres-ence of ZO-1 at the boundaries of human trophoblast cellsshortly after isolation (Supplemental Figure 1B, left pan-els, 12 hours at 20% O2), demonstrates that the cells arepolarized (64–66).
Our in vivo observation of increased Par6 expression at10–12 weeks gestation, when pO2 increases (opening ofintervillous space), appears to contrast our in vitro find-ings with isolated term CTs. However, during early pla-centation Par6 resides in different trophoblast cell layerscomprising the chorionic villi, and we speculate that reg-
ulation of Par6 by oxygen varies indifferent subsets of trophoblast cells(ie, CT, ST, and extravillous tropho-blasts). Indeed, preliminary evidencesuggests that increased Par6 levels invivo may be due to increased expres-sion of Par6 in extravillous tropho-blasts cells differentiating and mi-grating along the anchoringcolumn (data not shown). Hence, itis plausible that pO2 changes expe-rienced by the developing placentaact differentially on Par6 expres-sion in trophoblasts undergoing fu-sion vs those involved in migration.
Of clinical relevance, we report,for the first time, altered Par6 proteinexpression in preeclampsia. Becausehypoxia/oxidative stress character-izes PE placentas (18–21), the find-ing of increased Par6 expression is inaccord with our in vitro findingsdemonstrating that low oxygen up-regulates Par6 expression. Hence, inpreeclampsia, increased expressionof Par6 due to impaired oxygenationmay contribute to the altered tro-phoblast fusion characteristic of thispathology. Indeed, in PE placentas,Par6 localized to the trophoblasticlayer where it associates with ZO-1,specifically in CT cells.
In preeclampsia, trophoblast cellsmaintain an immature proliferative phenotype and exhibitaltered fusion (5, 23, 24, 38, 69). Thus, it is plausible thatthe increased Par6 levels in preeclampsia may prevent theloss of tight junctions from CT cell boundaries by main-taining trophoblast cells polarized, thereby preventingtheir fusion. Previous studies have demonstrated a de-crease in expression of two key regulators of fusion,GCM1 and syncytin 1, in PE placentas (23, 24). Subse-quent studies have demonstrated that the expression ofGCM1 and syncytin 1 are oxygen dependent; ie, expres-sion is decreased under hypoxic conditions through theinactivation of the phosphatidylinositol 3-kinase-Akt sig-naling pathway (70). The present observation of Par6 in-hibiting syncytin 1 expression after forskolin treatmentsuggests a role for Par6 in regulating syncytin 1 expres-sion. Whereas our studies focused on Par6 and the fuso-genic protein, syncytin 1, recent studies have identifiedanother fusogenic membrane retroviral envelop glycopro-tein termed “syncytin 2.” The involvement of Par6 in syn-
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Figure 7. Putative Model of Par6 Function in Trophoblast Fusion. During normal placentation,loss of Par6 promotes trophoblast fusion by inducing both loss of ZO-1 from tight junctions andcytoskeletal rearrangements. Under conditions of low oxygen, characteristic of PE placentas, Par6expression is increased, resulting in the permanent maintenance of tight junctions. Prevention ofdisruption of lateral adhesions between CT cells inhibits trophoblast fusion and impairssubsequent trophoblast turnover.
1306 Sivasubramaniyam et al Par6 and Trophoblast Cell Fusion Endocrinology, March 2013, 154(3):1296–1309
On the basis of our findings, we propose that the cellpolarity marker Par6 meets fusion during trophoblast dif-ferentiation (Figure 7). In order for trophoblast fusion toprogress, Par6 expression needs to be decreased to allowfor loss of cell adhesion and changes in cytoskeletal reor-ganization in trophoblast cells. However, in response tothe decreased O2 milieu in preeclampsia, Par6 expressionis increased which puts a brake on the fusogenic processand thereby contributes to the altered trophoblast fusioncharacteristic of this pathology.
Acknowledgments
We thank Dr. Dragica Curovic for placental collection and theBioBank Program of the Canadian Institutes of Health ResearchGroup in Development and Fetal Health (CIHR Grant MGC-13299), the Samuel Lunenfeld Research Institute, and the MountSinai Hospital Department of Obstetrics & Gynaecology forsome of the human specimens used in this study.
Address all correspondence and requests for reprints to:Isabella Caniggia, Mount Sinai Hospital, Samuel Lunenfeld Re-search Institute, 25 Orde Street, Room 6-1004-3, Toronto, On-tario, Canada M5T 3H7. E-mail: [email protected].
This work was supported by the Canadian Institutes ofHealth Research (CIHR) Grant (MOP-14096) (to I.C.). T.S. andM.M.-S. are supported by the Ontario Graduate Scholarship.I.C. is the recipient of a midcareer CIHR/Institute of Gender andHealth award from the Ontario Women’s Health Council.
Disclosure Summary: None of the authors has any competingfinancial interests in relation to the work described in the presentmanuscript or other conflict of interests.
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