Suppression of Mitochondrial Electron Transport Chain Function in the Hypoxic Human Placenta: A Role for miRNA-210 and Protein Synthesis Inhibition.

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by  Colleoni, Francesca

Suppression of Mitochondrial Electron Transport Chain
Function in the Hypoxic Human Placenta: A Role for
miRNA-210 and Protein Synthesis Inhibition
Francesca Colleoni1*, Nisha Padmanabhan1., Hong-wa Yung1., Erica D. Watson1, Irene Cetin2,
Martha C. Tissot van Patot1, Graham J. Burton1, Andrew J. Murray1
1Department of Physiology, Development & Neuroscience, and Centre for Trophoblast Research, University of Cambridge, Cambridge, United Kingdom, 2Unit of
ObstetricsandGynecology,DepartmentofClinicalSciences‘‘LuigiSacco’’,UniversityofMilan,Milan,Italy
Abstract
Fetal growth is critically dependent on energy metabolism in the placenta, which drives active exchange of nutrients.
Placentaloxygenlevelsarethereforevital,andchronichypoxiaduringpregnancyimpairsfetalgrowth.Herewetestedthe
hypothesis that placental hypoxia alters mitochondrial electron transport chain (ETS) function, and sought to identify
underlying mechanisms. We cultured human placental cells under different oxygen concentrations. Mitochondrial
respirationwasmeasured,alongsidelevelsofETScomplexes.Additionally,westudiedplacentasfromsea-leveland high-
altitude pregnancies. After 4 d at 1% O (1.01 KPa), complex I-supported respiration was 57% and 37% lower, in
2
trophoblast-likeJEG3cellsandfibroblasts,respectively,comparedwithcontrolsculturedat21%O (21.24 KPa);complexIV-
2
supportedrespirationwas22%and30%lower.Correspondingly,complexIlevelswere45%lowerinplacentasfromhigh-
altitude pregnancies than those from sea-level pregnancies. Expression ofHIF-responsive microRNA-210 was increased in
hypoxic fibroblasts and high-altitude placentas, whilst expression of its targets, iron-sulfur cluster scaffold (ISCU) and
cytochromecoxidaseassemblyprotein(COX10),decreased.Moreover,proteinsynthesis inhibition,afeatureofthehigh-
altitude placenta, also suppressed ETS complex protein levels. Our results demonstrate that mitochondrial function is
alteredinhypoxichumanplacentas,withspecificsuppressionofcomplexesIandIVcompromisingenergymetabolismand
potentially contributing to impairedfetal growth.
Citation:ColleoniF,PadmanabhanN,YungH-w,WatsonED,CetinI,etal.(2013)SuppressionofMitochondrialElectronTransportChainFunctionintheHypoxic
HumanPlacenta:ARoleformiRNA-210andProteinSynthesisInhibition.PLoSONE8(1):e55194.doi:10.1371/journal.pone.0055194
Editor:YidongBai,UniversityofTexasHealthScienceCenteratSanAntonio,UnitedStatesofAmerica
ReceivedAugust2,2012;AcceptedDecember19,2012;PublishedJanuary30,2013
Copyright:(cid:2)2013Colleonietal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:DrMurraythankstheResearchCouncilsUKforsupportinghisacademicfellowshipandProfBurtongratefullyacknowledgessupportforhisresearch
fromtheWellcomeTrust(084804/2/08/Z).ThisstudywasprimarilysupportedbyActionMedicalResearch,projectgrantnumberSP4545.Thefundershadnorole
instudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
.Theseauthorscontributedequallytothiswork.
Introduction however, fetal oxygen consumption at high-altitude, when
corrected for body weight, is not different to that at sea level
During the first trimester of pregnancy, the human fetus [7],thusoxygendeprivationperseisnotthoughttoberesponsible
develops in an environment characterised by a very low partial
fortheimpairment infetalgrowth.Instead,ithasbeenproposed
pressure of oxygen (pO ) [1], which is strikingly close to that
2 thatthehighaltitudeplacentaundergoesmetabolicremodellingto
experienced by mountaineers high on Mt Everest [2]. This
lower its own oxygen consumption, thereby maintaining oxygen
condition was termed Everest in utero, by Joseph Barcroft more
delivery to the fetus but at the cost of altered substrate delivery.
than 60yearsago, andisbelieved tofavour organogenesis inthe
This concept has been extensively reviewed by ourselves and
embryo, and cell proliferation and angiogenesis in the placenta
others [8–9],yettheunderlying mechanismsremain unresolved.
[1].
Placental dysfunction lies at the core of many common
At high altitude, where women are exposed to atmospheric
complicationsofpregnancy,suchasintrauterinegrowthrestriction
hypobarichypoxia,thehypoxicnatureoftheuterineenvironment
and pre-eclampsia. These disorders can jeopardise the health of
is further exacerbated.Such chronic exposure to hypobaric both mother and fetus, accounting for ,60% of babies weighing
hypoxia during pregnancy leads to babies that are small for
less than 1000g that survive to only one year of life [10]. The
gestational age [3], and an increased incidence of intrauterine
pathophysiology of pre-eclampsia is not completely understood,
growth restriction and pre-eclampsia [4,5]. Curiously, normal
but is thought to result from incomplete remodelling of the
oxygen delivery to the fetus is maintained at altitudes of around
maternal spiral arteries [11], disrupting the normal flow of blood
3000mabovesea-leveldespitealowerarterialpartialpressureof
into the placenta and risking ischemia/reperfusion injury [1].
oxygen,atleastinpartduetoincreasederythropoiesisinboththe
Indeed, the induction of oxidative stress is a component of pre-
maternal and fetal circulations [6]. Somewhat paradoxically,
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HypoxiaandHumanPlacentalMitochondria
eclampsia [12], supported by reports of increased pro-oxidant whether protein synthesis inhibition suppresses mitochondrial
factors [13,14]anddecreased anti-oxidant defences [14]. function,westudiedthesameplacentalcellswhenculturedat21%
Inthisregard,placentalmitochondriaarelikelytoplayacentral O in the presence of a non-lethal dose of salubrinal, a
2
role in pre-eclampsia, being producers of reactive oxygen species phosphatase inhibitor which prevents dephosphorylation of
(ROS) at complexes I and III of the electron transport system eukaryotic initiation factor 2 subunit a (eIF2a). Finally, we
(ETS), and themselves targets of oxidative stress. Increased extended our investigation into human placentas from high-
superoxide production has been reported in pre-eclamptic altitudepregnancies,comparingexpressionlevelsofmitochondrial
placentas [15], suggesting that the mitochondria are at increased proteins,miR-210anditstargetswiththoseinplacentasfromsea-
risk of oxidative damage. Indeed, in other metabolically-active level pregnancies. We hypothesized that overlapping, cell-specific
tissues, such as cardiac and skeletal muscle, oxidative stress is mechanisms drive modifications of ETS activity in the hypoxic
associatedwithprofoundlyalteredmitochondrialfunction[16,17]. placenta tosuppress oxidative metabolism.
For example, in hypoxic skeletal muscle, the downregulation of
ETS complexes I and IV may be an adaptive response to Materials and Methods
respectivelylimitROSproductionandoxygenconsumption[18].
Additionally,recentdatasuggestthatinearly-onsetpre-eclampsia Ethics Statement
there is a high incidence of endoplasmic reticulum (ER) stress, a Informed, written consent for the use of placental samples to
phenomenon strongly associated with oxidative stress and which research adaptations to hypoxia was obtained from subjects
shares a similar etiology[19]. recruitedatSt.Vincent’sGeneralHospitalinLeadville,CO,USA
Stabilization of hypoxia-inducible factor-1a (HIF-1a) under (3,100 m above sea-level) with the approval of the Colorado
hypoxic conditions leads to a downregulation of mitochondrial Multiple Institutional Review Board (COMIRB Protocol 00–
oxygenconsumption[20,21],andtheHIF-responsivemicroRNA- 623),and from subjects recruited at the University College
210 (miR-210) has been strongly implicated in this response Hospital, London, UK (sea-level), with the approval of The
[22,23]. MiR-210 represses the iron-sulfur complex assembly UniversityCollegeLondonHospitalsCommitteeontheEthicsof
proteins (ISCU1/2) [23], which are required for the correct HumanResearch(theJointUCL/UCLHEthicsCommittee,Ref
assemblyofiron-sulfurclustersinETScomplexesI,IIandIII.It No:03/0135).
alsorepressesthecytochromecoxidaseassemblyprotein(COX10)
[24],whichisessential forassembly of ETScomplexesI andIV. Chemicals and Reagents
HIF-mediated induction of miR-210 is, therefore, a potential All chemicals and tissue culture reagents were purchased from
mechanism underlying placental remodelling in the oxidatively- Sigma-Aldrich and Invitrogen Ltd (Paisley, UK) respectively,
stressed high-altitude placenta, and is known to be elevated in except where otherwise mentioned. Salubrinal was purchased
placental tissue derived from pre-eclamptic patients [25,26,27], from Chem-Bridge Corporation (San Diego, USA). The anti-Hu
and in a recent study was shown to regulate trophoblast TotalOxPhosComplexprimaryantibodykitwaspurchasedfrom
mitochondrial respiration in pre-eclampsia [27]. An alternative Invitrogen, anti-ISCU1/2 (FL-142) from Santa Cruz Biotechnol-
mechanism,however,mayresultfromproteinsynthesisinhibition, ogy (Insight Biotechnology, Wembley, UK), anti-COX10 from
since there is marked evidence of ER stress resulting in protein Proteintech (Manchester, UK) and anti-citrate synthase from
synthesis inhibition in high-altitude placentas [28], a feature AlphaDiagnostics (SanAntonio,TX, USA).
shared with the pre-eclamptic placenta [19]. Protein synthesis
inhibition might therefore restrict the synthesis of ETS complex Cell Cultures
subunits, furtherrepressing oxidative metabolismat theplacenta. PrimaryhumanplacentalfibroblastswereagiftfromProfessor
In this study, we aimed to determine the effects of chronic Ashley Moffett(UniversityofCambridge) andwereisolatedfrom
hypoxiaonmitochondrialfunctioninthehumanplacentaandthe first and early second trimester placentas with Local Ethical
underlying mechanisms. We investigated mitochondrial respira- Committee approval [28]. These cells were grown in Dulbecco’s
tionandmRNAandproteinexpressionofETScomplexesintwo Modified Eagle medium (DMEM), supplemented with 5% HI-
placental cell types grown at different oxygen tensions; a human FBS,penicillin(100 U/ml),streptomycin(100 mg/ml),at37uCin
trophoblast-likecelllineandprimaryhumanplacentalfibroblasts. a 5% carbon dioxide (CO ) atmosphere. JEG-3 cells were a gift
2
Volumetrically, these cells represent the principal components of from Professor Ashley Moffett (University of Cambridge), and
the placenta, but may have different metabolic properties due to were originally purchased from the American Type Culture
their different functions. JEG3 cells possess many biological and Collectionandculturedaccordingtotheirinstructions(ATCC,cat
biochemical characteristics of syncytiotrophoblasts [29], they no. HTB-144 and HTB-36) [28,30]. JEG3 cells were grown in
produce placental hormones and express enzymes involved in RPMI1640mediumsupplementedwith5%heat-inactivatedFBS
steroidogenesis. They have been widely used to examine the (HI-FBS),penicillin(100U/ml),andstreptomycin(100 mg/ml)at
hormonal function of trophoblast cells and intracellular receptor 37uCina5%CO atmosphere.Thehumanchoriocarcinomacell
2
mechanisms,andasamodelinagreatnumberofstudiesdevoted line BeWo cells were a gift from Dr. Stephen Charnock-Jones
to the effects of endocrine-active compounds (xenoestrogens, (University of Cambridge) (ATCC, cat no. CCL-98). [28]. They
fitoestrogens, dioxins and pesticides) [29]. We also carried out were cultured in DMEM/F12 medium supplemented with 10%
supporting experiments in cultures of the additional, trophoblast- HI-FBS,penicillin(100 U/ml),streptomycin(100mg/ml),at37uC
likeBeWocellline.Meanwhilefibroblasts,inadditiontobeingthe ina 5%carbon dioxide(CO ) atmosphere.
2
mostnumerouscelltypeintheplacenta,performthefunctionsof For hypoxia experiments, placental fibroblasts, JEG3 cells and
stromal biosynthesis and structural support, and are therefore BeWocellswereseededatalowdensitywithfreshmedia(RPMI
essential to the growth of the villous trees, itself a metabolically- 1640 medium, DMEM and DMEM/F12, respectively) in the
demanding process. presenceofHI-FBS,penicillinandstreptomycinandwereplaced
Furthermore, we measured the expression of miR-210 and its directly into humidified hypoxic chambers (Ex Vivo system,
downstream targets, to determine whether they could be Biospherix Ltd, NY, USA) containing 1% O (1.01KPa)/5%
2
implicated in a potential mechanism. Moreover, to investigate CO balancedinnitrogenfor4days.Controlcellswereincubated
2
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HypoxiaandHumanPlacentalMitochondria
at 21% O (21.24KPa)/5% CO or 10% O (10.11 KPa)/5% malate were added to the chambers, and complex I-supported
2 2 2
CO under standard culture conditions. A concentration of 21% state 2 respiration was recorded. State 3 respiration was
2
O was chosen as a first control as this is the normal O stimulated by the addition of 2 mM ADP. Next, complex I
2 2
concentrationtowhichJEG3andBeWocellshaveadapted,whilst was inhibited by the addition of 0.5 mM rotenone, before
10%O wasusedtomimicintraplacentalconditions[1,10]atthe 10mM succinate was added and complex II-supported state 3
2
start of the second trimester invivo. For hypoxic conditions, we respiration recorded. Electron transport was then inhibited at
selected 1% O2 as this was shown to elicit a hypoxic response in complex III by addition of 5 mM antimycin. Complex IV-
pilot studies. After 4 d, cells were harvested for analysis of supported state 3 respiration was stimulated by addition of
mitochondrialrespiration,protein,mRNAandmiRNAexpression 0.5 mM TMPD and 2 mM ascorbate. Between experiments,
levels or mitochondrial DNA (mtDNA) concentrations. After 4 oxygen electrode chambers were washed for at least 40min
days, hypoxia (1% O2) decreases the proliferation of JEG3 cells, with 100% ethanol and then several times with water to remove
BeWo cells and placental fibroblasts by 40%, 60% and 18%, any trace of respiratory inhibitors.
respectively [28].
For protein synthesis inhibition experiments, placental fibro- Western Blotting
blasts and JEG3 cells were seeded at a low density with fresh
Cultured cells were harvested and washed with ice-cold PBS,
media as before. Untreated control cells and cells grown with a
before being scraped into cell lysis buffer (20mM Tris, 150mM
sublethaldoseofsalubrinal(17.5 mM)wereincubatedat21%O /
2 NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5mM
5% CO for 3 d under standard conditions, and harvested for
2 sodium pyrophosphate, 1 mM glycerolphosphate, 1 mM
analysisofproteinlevels.Inhibitionofproteinsynthesisusingthese
Na VO , and complete mini protease inhibitor cocktail (Roche
culturedconditionswasrecentlyconfirmedinastudypublishedby 3 4
Diagnostics, East Sussex, UK, pH 7.5), and transferred to a
ourgroup[28].BeWocellswereseededatalowdensitywithfresh
microfugetube.Afterpipettingupanddown,30times,cellswere
media as before. Untreated control cells and cells grown with a
kept on ice for 20min with occasional vortexing and centrifuged
sublethal dosage of tunicamycin (2.5 ug/ml) or thapsigargin
at10,000 gfor5min.Placentalsampleswerehomogenisedinthe
(0.4 mM ) were incubated at 21% O /5% CO for 3 d under
2 2 samelysisbufferasthecellsusinglysingmatrixtubes(typeD,MP
standard conditions, and harvested for analysis of ETS protein
Biomedicals).
levels.
Bicinchoninic Acid (BCA) was used to determine protein
concentrations in the cultured cell and tissue lysates. Equal
Tissue Collection from Human Subjects
amounts of protein were resolved using SDS-PAGE and
Exclusion criteria for subjects included renal disease, cardiac
transferred to nitrocellulose membrane. Western blotting analysis
disease, diabetes, chronic hypertension, pregnancy-induced
of protein expression was performed as described previously [33]
hypertension, pre-term delivery or any complication of preg-
with Ponceau red staining used to normalize for protein-loading.
nancy. Placentas (n=6) were collected at sea level from elective
After incubation with primary and secondary antibodies, en-
non-labored caesarean deliveries (two tissue samples from
hanced chemiluminescence (ECL) (GE Healthcare, Little Chal-
different regions of each placenta), whilst 3 placentas (four
font,UK)andX-rayfilm(Kodak,Hempstead,UK)wereusedto
tissue samples from different regions of each placenta) was
detect the bands. Band intensity was quantified by ImageJ (U.S.
collected at 3100m altitude, again from elective non-labored
NationalInstitutes ofHealth, Bethesda, MD,USA).
caesarean deliveries. Since the labor process is a strong inducer
of oxidative stress [31], only samples delivered by caesarean
DNAandRNAExtractionandQuantitativeReal-timePCR
section were used in this study. The samples were collected
Analysis
immediately after delivery, by the same team at each site to
eliminate differences in tissue handling. Each placenta was DNAandRNAwereextractedfromculturedcellsandhuman
weighed and samples were taken using a systematic random placentalsamplesusingSIGMAGenEluteTMMammalianGeno-
system by which each placenta was divided into five areas. Two mic DNA Miniprep Kit and QIAGEN RNAeasy Mini kit,
full-thickness samples were taken from each area. Samples were respectively according to the manufacturer’s instructions. RNA
washed in phosphate buffered saline (PBS) to remove blood, was further treated with TURBO DNase (Ambion) to eliminate
snap-frozen in liquid nitrogen within 10min of delivery and DNA contamination. A QIAGEN MiRNeasy Mini Kit was used
stored at 280uC until further analysis. toextract miRNAs.
For total RNA analysis, cDNA was prepared using the first
Mitochondrial Respirometry strand synthesis kit from Fermentas. Real-time PCR was
Mitochondrialrespirationwasmeasuredinpermeabilisedcells, performed using SYBR green Master Mix (Eurogentech) on ABI
asdescribedpreviously[1,32].Briefly,harvestedcellswerewashed PRISM7500SequenceDetectionSystem.Sampleswereanalysed
with PBS and re-suspended in respiratory medium (0.5mM in triplicate and expression levels were normalised to the
EGTA, 3mM MgCl .6H O, 20mM taurine, 10mM KH PO , housekeeping gene hypoxanthine guanine phosphoribosyl trans-
2 2 2 4
20mM HEPES, 1 mg/ml BSA, 60mM potassium-lactobionate, ferase (HPRT) or to HPRT and b-actin, together. Melting curve
110mMmannitol,0.3mMdithiothreitol,pH 7.1).Densityofcell analysiswasperformedtoensurespecificityofPCRproducts.Fold
suspensions was determined using a haemocytometer and 26106 change was calculated using a standard curve method. Primer
cells wereadded toafinal volume of500mlrespiratory medium, sequences are available upon request.
equilibrated to atmospheric O in a water-jacketed oxygen For microRNA analysis, cDNA was prepared using RevertAid
2,
electrode chamber at 37uC (Strathkelvin Instruments Ltd, H Minus Reverse Transcriptase (Fermentas) and microRNA
Glasgow, UK) and the chamber was sealed. Cell membranes specific RT primers (TaqMan MiRNA assay). Real-time PCR of
wereselectivelypermeabilisedwithdigitonin(50mg/ml)for5 min, miRNA targets was performed using TaqMan MicroRNA assays
before mitochondrial respirationwas measured. (hsa-miR-210) according to manufacturer’s instructions and
A substrate/inhibitor titration was used to analyse ETS normalised to a reference miRNA (RNU48). Fold change was
complexes I, II and IV. Initially, 10mM glutamate and 5mM calculated usingDDCt method.
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HypoxiaandHumanPlacentalMitochondria
Real-time Quantitative PCR Analysis for Mitochondrial 33% lower compared with cells at 21% O (p,0.01), and 22%
2
DNA Content lower than cells at 10% O2 (p,0.05) (Figure 1B). Complex I-
Total DNA was extracted using QIAamp DNA Mini kit Q supported state 3 rates were 56% and 52% lower in JEG3 cells
(Qiagen, Milan, Italy). ABI Prism 7500 Sequence Detection cultured at 1% O2 compared with cells cultured at 21% O2
System was used for real-time quantitative polymerase chain (p,0.05) and 10% O2 (p,0.05), respectively (Figure 1B) once
reaction(PCR)analysisusingtheRnasePgene,asanendogenous again, there was no significant difference in RCRs between
control and cytochrome b as mitochondrial target gene. Samples difference culture conditions, suggesting no alteration in proton
were analysed in triplicate. The D cycle threshold (DCt) values leak.Similartoourobservationinfibroblasts,nodifferenceswere
fromeachsamplewereobtainedbysubtractingthevaluesforthe apparentwithrespecttocomplexII-supported state 3respiration
reference gene from the sample Ct, thus normalizing to nuclear rates in JEG3 cells cultured under all three oxygen conditions
DNA. (Figure 1B). However, complex IV-supported state 3 respiration
rates were 22% and 23% lower in cells cultured at 1% O
2
Statistics compared with cells cultured at 21% (p,0.001) and 10% O2,
Resultsareexpressedasmeans6SEM.Alldatawerechecked respectively(p,0.001)(Figure1B).Together,thesedataindicatea
for normal distribution. Analysis of variance (one way ANOVA) strikingly similar response of lowered mitochondrial respiration
with repeated measures and least significant difference (LSD) post rates under severe hypoxia in two different placental cell types.
hocindependentunpairedttestswereusedtodeterminedifferences Supporting experiments were also performed on another chorio-
between groups in different oxygen environments for JEG3, carcinoma cell line, BeWo cells, and demonstrated that hypoxia
fibroblasts and BeWo cells (for respirometry, protein and DNA impaired Complexes I–II and IV-supported state 3 rates (Figure
levels). Independent t tests were used to determine differences S1A).
between placental samples from different altitudes and for JEG3
and fibroblasts mRNA quantification experiments. Data were Hypoxia Alters the Expression of Electron Transport
considered statisticallysignificant at p,0.05. Chain Complexes in Placental Cells
Since the mitochondrial respiratory rates through complex I
Results were lowered in hypoxic conditions, we sought to determine
whetherthiswasduetochangesintheexpressionofcomponents
Mitochondrial Respiration Rates are Lower in Hypoxic
thatmakeuptheETSmachinery.DecreasedcomplexI-supported
Placental Cells
rates can result from a decrease in mitochondrial membrane
To investigate whether mitochondrial function was altered in
potential, since many substrates for complexI rely on theproton
hypoxic placental cells, we first compared respiration in primary
gradientforimport.First,weusedqRT-PCRtodeterminemRNA
human placentalfibroblasts culturedunder21%O andhypoxic
2 expressionlevelsofNDUFB8(NADHdehydrogenase(ubiquinone)
(1% O ) conditions. In order to study responses across a broad
2 1 beta subcomplex 8; encodes a complex I subunit), mtCOX2
spectrum of oxygen concentrations, we used three different
(mitochondriallyencodedcytochromecoxidaseII,alsoknownas
conditions: 1% O 10% O and 21% O . All state 2 and state
2, 2 2 MTCO2 or COX2; encodes a complex IV subunit) and ATP5a
3 rates were the same between 21% and 10% O culture
2 (ATP-synthase5a;encodesacomplexVsubunit)inbothplacental
conditions indicating that these concentrations could be used as
fibroblastandJEG3cellsculturedinhypoxicconditionscompared
ourcontrols(Figure1A).Inhypoxicconditions,at1%O ,state2
2 tothoseculturedinnormoxia.TranscriptlevelsofNDUFB8were
respiration rates of fibroblasts were 56% lower compared with
unchanged in both fibroblasts and JEG3 irrespective of O
thoseculturedat21%O (56%ofcontrolrates;p,0.01)and47% 2
2 concentration (Figures 1C and 1D). However, mRNA expression
lower compared to 10% O (Figure 1A). Complex I-supported
2 levelsofmtCOX2were37%(p,0.05)and44%(p=0.07)lowerin
state 3 rates were 43% and 36% lower in fibroblasts cultured at
fibroblastsculturedat1%O comparedtofibroblastsat10%O
1% O compared with those cultured at 10% O (p,0.05) and 2 2
2 2 and21%O ,respectively(Figure1C).ATP5A(complexV)mRNA
21% O , respectively (Figure 1A), and there was no significant 2
2 expression was also significantly reduced in fibroblasts grown at
difference in respiratory control ratios (RCRs; state 3/state 2)
1%O comparedto21%O (49%ofcontrol;p,0.05)butagain
between difference culture conditions, suggesting no alteration in 2 2
did not change in JEG3. Whilst there were changes in mRNA
proton leak. Complex II-supported state 3 respiratory rates were
expressioninfibroblasts,therewereclearlynonepresentinJEG3
thesameinfibroblastsculturedunderallthreeoxygenconditions
cells.
(Figure 1A). However, complex IV-supported state 3 rates were
29% and 24% lower in fibroblasts cultured at 1% O (p,0.01) Therefore, to determine whether a post-transcriptional mech-
2
compared with those cultured at 21% and 10% O , respectively anism was operating to regulate the protein subunits of the ETS
2
(Figure 1A). complexes I–IV (NDUFB8, subunit 30KDa, subunit core2 and
Primary trophoblast cells isolated from human placentas were mtCOX2, respectively) and V (ATP5a) in hypoxia, we used
not suitable for these metabolic studies as they display molecular western blot analysis to quantify the expression of these proteins.
evidenceofERstress(datanotshown).Thismayaccountforthe Similar to respiratory rates, protein levels of all subunits tested
factthatmitochondrialmembranepotentialdeclinesprogressively were unchanged in placental fibroblasts and JEG3 cultured at
in culture, to the point that it is almost totally lost at 96h [34]. 21% O2 and 10% O2. Although protein levels of the complex I
Overthesameperiodthereisactivationofcaspases3and9,and subunit (NDUFB8) in fibroblasts at 1% O2 was 66% lower
evidenceofapoptoticcelldeath.Therefore,weanalyzedahuman comparedto21%O2and72%lowercomparedto10%O2,these
placental trophoblast-like cell line (JEG3) to determine whether values did not reach statistical significance (Figure 1E). Interest-
the hypoxia-induced mitochondrial respiratory rate changes we ingly,mtCOX2,theproteinsubunitofcomplexIVshoweda70%
observedintheplacentalfibroblastswerecelltype-specific.Aswith decreaseinexpressioninfibroblastsculturedat1%O compared
2
primaryhumanplacentalfibroblasts,therewerenodifferencesin with fibroblasts cultured at 21% O (p,0.05) and 10% O
2 2
all respiratory rates between cells cultured at 21% and 10%. In (p,0.05) (Figure 1E). This was consistent with a decrease in
JEG3 cells cultured at 1% O , however, respiration rates were mtCOX2 mRNAexpression inthese cells.
2
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HypoxiaandHumanPlacentalMitochondria
Figure 1. Mitochondrial function and ETS mRNA and protein expression were altered in fibroblasts and JEG3 cells cultured in
hypoxicconditions.A,B)State2andstate3respirationrateswiththecomplexIsubstrates,glutamateandmalate;andstate3respirationrates
withthecomplexIIsubstrate,succinate,andcomplexIVsubstrates,TMPDandascorbatein A)fibroblastsandB)JEG3.C,D)TranscriptlevelsofETS
complexesI,IVandV(ATP-synthase)inC)fibroblastsandD)JEG3.E,F)ProteinlevelsofETScomplexesI-IVandV(ATP-synthase)inE)fibroblasts
andF)JEG3.*p,0.05,**p,0.01,***p,0.001comparedwithcellsculturedat21%O2;{p,0.05,{{p,0.01,{{{p,0.001comparedwithcells
culturedat10%O2.Threeindependentexperimentswereperformedinduplicateforeachcondition;eachexperimentwascarriedoutinduplicate.
doi:10.1371/journal.pone.0055194.g001
Similar to the fibroblast cells, levels of complex I subunit suggestthatspecificcomponentsoftheETScomplexesareeither
NDUFB8was,64%and55%lowerinJEG3cellsculturedat1% transcriptionally orpost-transcriptionally regulatedin responseto
O thanincellsculturedat21%(p,0.01)and10%O (p,0.05), hypoxia and that this process may occur in a placental cell-type
2 2
respectively (Figure 1F). The concentration of the complex IV specific manner. To exclude the possibility that a change in
protein subunit (mtCOX2) was also decreased, being 64% and mitochondrial content might underlie these observations, we
57% lower in JEG3 cells cultured at 1% O2 compared with cells measured mitochondrialDNA content andcitratesynthase levels
cultured at 21% (p,0.01) and 10% O2 (p,0.01), respectively in both fibroblasts and JEG3 cells in all oxygen conditions and
(Figure 1F). Since the mRNA expression of these genes was foundno significant differences (Figures2A–D).
unchanged (Figure 1D), the reduction in the levels of these
proteinslikelyreflectspost-transcriptionalregulation.Additionally,
Expression of microRNA-210 and its Targets is Altered in
protein levels of complex III subunit (sub core 2) were 27% and
Hypoxic Placental Fibroblasts
26% lower in JEG3 cells cultured at 1% O than in control cells
2 One method of post-transcriptional regulation involves miR-
cultured at 21% (p,0.05) and 10% O (p,0.05), respectively
2 NAs,whichare23nucleotideRNAsthatbindtocomplementary
(Figure 1F); while this trend was not significant in fibroblasts
sequences on mRNA transcripts and cause gene silencing by
(Figure1E).Lastly,complexVsubunit(ATP-synthase5a)showed
translationalrepressionormRNAdegradation[35].Followingour
26% lower levels of protein in JEG3 cells cultured at 1% O
2 finding that complex I and complex IV protein subunits were
compared with cells at 21% O (p,0.05) but not different from
2 decreased in both placental cell types in response to hypoxic
cellsculturedat10%O (Figure1F).ThecomplexIIsubunit(sub
2 conditions, we investigated the expression of miRNA-210, a
30KDa) concentration was not different in fibroblasts and JEG3
knownregulatorofthesecomplexes,alongwiththeexpressionof
cellsculturedunderallconditions(Figures1Eand1F).Incontrast,
its targets COX10 (cytochrome c oxidase assembly protein; a
but in agreement withrespirometry data, in BeWo cells,hypoxia
subunit of complexIV) and ISCU1/2 (iron-sulfur cluster scaffold
resulted in a downregulation of representative subunits of all five
proteins),todeterminewhetherthemiRNApathwaymightplaya
mitochondrial complexes (Figure S1B). Altogether, these data
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Figure2.CitratesynthaseproteinlevelsandmitochondrialDNAcopynumberinplacentalfibroblastsandJEG3cells.A,B)Protein
levelsofcitratesynthaseinA)fibroblastsandinB)JEG3cells.C,D)MitochondrialDNAcontentinC)fibroblastsandD)JEG3.Minimumoffour
biologicalreplicatespercelltypeforeachconditionwereperformed.
doi:10.1371/journal.pone.0055194.g002
mechanistic role in this context. Remarkably, exposure of mRNA expression, protein levels of COX10 and ISCU1/2 were
placental fibroblasts to 1% O resulted in a substantial increase significantly lower in JEG3 cells exposed to 1% O compared to
2 2
inmiR-210levels,12.8and9.7foldgreaterthanfibroblastsgrown those at 21% O (p,0.01 and p,0.05, respectively). These
2
at 21% O (p,0.001) and 10% O (p=0.001), respectively values were also lower than in cells grown at 10% (p,0.05;
2 2
(Figure 3A). This increase in miR-210 in hypoxic fibroblasts also Figure 3F). Altogether, these findings suggest that different
corresponded with a decrease in mRNA and protein levels of its placental cell types utilise distinct mechanisms to regulate
targets COX10 (p=0.07; 1% O vs. 10% O ) and ISCU1/2 mitochondrial function: hypoxic fibroblasts initiate a miR-210-
2 2
(p,0.01;1%O vs.both10%O and21%O )(Figures3C,3E). based process whereas trophoblast-like JEG3 cells may use an
2 2 2
Alternatively, in JEG3 cells, there was no change in miR-210 alternative mechanism.
expression under hypoxic conditions (Figure 3B). Yet, we
observed an increase in mRNA levels of both COX10 Protein Synthesis Inhibition Results in Lower Levels of
(p,0.01, 1% O2 vs. 21% O2; p,0.05, 1% O2 vs. 10% O2) ETS Proteins in Placental Cells
and ISCU1/2 (p,0.05, 1% O2 vs. both 10% O2 and 21% O2) NDUFB8 transcript levels did not change (Figures 1C and
in hypoxic JEG3 cells (Figure 3D). Despite this increase in
1D) however protein levels of NDUFB8 were over 60% lower
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HypoxiaandHumanPlacentalMitochondria
Figure3.MicroRNA-210expressionisinducedinhypoxicfibroblastsbutnotinhypoxicJEG3cells;RNAandproteinexpressionof
COX10andISCU1/2aredecreasedinbothfibroblastsandJEG3cellsculturedinhypoxiccondition(1%O2).A,B)MiR-210expression
inA)fibroblastsandinB)JEG3.C,D)TranscriptlevelsofCOX10andISCU1/2inC)fibroblastsandD)JEG3.E,F) ProteinlevelsofCOX10andISCU1/
2inE) fibroblastsandinF)JEG3.*p,0.05,**p,0.01,***p,0.001comparedwithcellsculturedat21%O2;{p,0.05comparedwithcellscultured
at10%O2.Minimumofthreebiologicalreplicatespercelltypeforeachconditionwereperformed.
doi:10.1371/journal.pone.0055194.g003
in both cell types under hypoxia, indicating that other Interestingly,amorerobustchangewasobservedinJEG3cells
mechanism(s) regulate the protein expression. To investigate culturedwithsalubrinalincludingadecreaseinproteinexpression
this further, we cultured primary placental fibroblasts and JEG3 of all ETS complexes assessed. Similar to placental fibroblasts,
cells in normoxia in the presence and absence of 17.5mM protein levels ofcomplexesI (NDUFB8) andIV(mtCOX2) were
salubrinal, an inhibitor of the protein synthesis initiation factor 97%and75%lowerinJEG3cellsthanincontrols(p,0.0001and
eIF2a. Salubrinal did not alter protein levels of representative p,0.001,respectively)(Figure3B).However,analysisofcomplex-
subunits from ETS complexes II (sub 30KDa), III (sub core 2) esII(sub30KDa),III(subcore2)andV(ATP5a)alsorevealeda
and V (ATP-synthase) in primary placental fibroblasts. Howev- significantrepressionofproteinexpressioninJEG3cellscompared
er, complex I (NDUFB8) and complex IV (mtCOX2) protein to controls by 31% (p,0.01), 80% (p,0.05) and 34% (p,0.01),
expression was depressed by 88% (p=0.06) and 71% (p,0.01), respectively (Figure 4B).
respectively, in fibroblasts cultured with salubrinal relative to Tofurthersupporttheideathatthereisalinkbetweenprotein
those cultured without salubrinal (Figure 4A). synthesis inhibition and levels of the ETS complexes, we carried
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HypoxiaandHumanPlacentalMitochondria
Figure4.SublethaldosageofsalubrinaldownregulatesETS,COX10andISCU1/2proteinlevelsinfibroblastsandJEG3cells.A,B)
ProteinlevelsofETScomplexesI-IVandV(ATP-synthase)inA)fibroblastsandB)JEG3.C,D)ProteinlevelsofCOX10andISCU1/2inC)fibroblasts
andinD)JEG3.*p,0.05,**p,0.01,***p,0.001comparedwithcellsculturedwithoutsalubrinal.Minimumofthreebiologicalreplicatespercell
typeforeachconditionwereperformed.
doi:10.1371/journal.pone.0055194.g004
outsupportingexperimentsonBeWocellsusingotherinducersof function in hypoxia, therefore we examined whether similar
ERstress:tunicamicinandthapsigargin.Tunicamycininducesthe effects occurred invivo using placentas from high-altitude
unfolded-proteinresponseandthapsigarginelevatescytosolicCa2+. pregnancies in which a high level of phosphorylation of eIF2a
Both molecules therefore induce ER stress, via different mecha- has been recently identified [28]. Sea-level and high-altitude
nisms, andin BeWo cells,bothinhibitorsaltered protein levels of placentasusedinthisinvestigationwerematchedformaternalage
representativesubunitsfromETScomplexes(FigureS2A). and gestational age and were non-labored, caesarean deliveries.
We also assessed whether salubrinal affected the expression of Birth weights were 364g lower at high altitude than at sea level,
COX10 and ISCU1/2 in fibroblasts and JEG3 cells. Salubrinal andsimilartootherstudieswithatrendtowardssmallerplacental
treatment did not alter COX10 protein levels in fibroblasts weights (Table1).
(Figure 4C), but levels were 55% lower in JEG3 cells (p,0.01) There were no differences in mRNA transcript levels of
(Figure 3D). Salubrinal decreased ISCU1/2 protein levels by 7% NDUFB8(complexI),mtCOX2(complexIV)andATP5A(complex
inplacentalfibroblasts(p,0.05)and30%inJEG3cells(p,0.01) V) in high-altitude placentas compared with sea-level controls as
(Figures 4C and 4D). Taken together, these data indicate that revealedbyqPCRanalysis(Figure–5A).Despitethis,proteinlevels
protein synthesis inhibition alone can have similar effects to of NDUFB8 were 48% lower in high-altitude placentas than in
hypoxia, in changing the expression of key proteins required for sea-level placentas (p,0.01). Furthermore, protein expression of
ETS function. However, placental fibroblast cells appear to be complexes II (sub 30KDa), III (sub core 2) and IV (mtCOX2)
more resistant to the effects of salubrinal and hypoxia than werelowerinhigh-altitudeplacentasthaninthosefromsea-level
trophoblast-like cells. pregnancies,by36%(p,0.01),28%(p,0.01)and32%(p,0.05),
respectively.ProteinlevelsofcomplexV(ATP5a)were,however,
High-altitude Pregnancy is Associated with Increased unchanged(Figure5B).Theseobservationsarereminiscentofthe
Expression of miR-210 changes seen in salubrinal-treated JEG3 cells suggesting that the
proteinsynthesisinhibition,previouslyreportedintheseplacentas
As our in vitro data suggests protein synthesis inhibition and
[28] maybeactingtosuppress mitochondrialrespiration.
miR-210-driven mechanisms likely regulate mitochondrial
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HypoxiaandHumanPlacentalMitochondria
Table1. Clinicalcharacteristics ofsea-leveland high-altitudepregnancies;placental samplesfrom non-labored,caesarean
deliveries were usedforETS complex protein, mRNAand microRNAanalysesin thisstudy.
PlacentasfromNon-LaboredCaesareanDeliveries
Sea-Level(n=6) High-Altitude(n=3)
MaternalAge(yrs) 31.562.1 34.061.0
GestationalAge(wks) 39.061.4 39.961.2
BirthWeight(g) 36276272 32636502
PlacentalWeightatBirth(g) 545644 530635
doi:10.1371/journal.pone.0055194.t001
The expression of miR-210 was 1.8-fold higher in placental translationalregulationofETSproteinsinhighaltitudeplacentas
samples from high-altitude pregnancies compared to sea-level andin placental cellscultured underhypoxicconditions.
placentas(p=0.06;Figure5D),whilstthemRNAexpressionofits
downstream target gene COX10 was 27% lower (p,0.01), and Discussion
COX10 protein expression was 30% lower (p,0.05) (Figure 5E
and 5F). No difference was observed in ISCU1/2mRNA Inallmetabolically-activetissues,sustainedhypoxianecessitates
appropriateresponsestomaintainenergeticandredoxhomeosta-
expression between the two types of placentas assessed
sis, and thereby supporting normal cellular function [16]. The
(Figure 5E). However, protein expression of ISCU1/2 was 21%
hypoxic placenta, however, faces the unique challenge of
lower (p=0.06), (Figure 5F), suggesting that miR-210 is likely
sustaining sufficient oxygen transfer to the circulation of the
involved in post-transcriptional repression of ISCU1/2. Overall,
developing fetus whilst meeting the oxygen demands of its own
these data point to overlapping mechanisms that modify the
metabolism [9]. The consequences of a dysfunctional response
Figure5.InductionofmiR-210,downregulationofETS,COX10andISCU1/2proteinlevelsofhigh-altitudehumanplacentas.A)
TranscriptlevelsofETScomplexesI,IVandV(ATP-synthase)andB)proteinlevelsofETScomplexesI-IVandV(ATP-synthase).C)MiR-210expression
insea-levelandhigh-altitudeplacentas.D)TranscriptlevelsandE)proteinlevelsofCOX10andISCU1/2.F)Proteinlevelsofcitratesynthase.*p,0.05
comparedwithsea-levelplacentas.Placentas(n=6)atsealevel(twotissuesamplesfromdifferentregionsofeachplacenta),and3placentasat
3100maltitude(fourtissuesamplesfromdifferentregionsofeachplacenta)wereperformed.Eachsamplewascarriedoutatleastinduplicate.
doi:10.1371/journal.pone.0055194.g005
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couldeitherresultinfetaloxygendeprivation,ifplacentaloxygen Supporting a post-transcriptional regulatory mechanism, we
consumption remained too high, or fetal nutrient deprivation, if observedastrongupregulationofmiR-210infibroblastsfollowing
energetic impairment in the placenta limited the active transport hypoxia, perhaps reinforcing the idea of mitochondrial suppres-
of substrates. A careful partitioning of oxygen between these sion playing a protective role in hypoxia [23,44,45]. No such
competing demands is therefore vital to prevent fetal growth changes were noted in JEG3 cells, however, though it has been
restriction and limit oxidative damage in placental and fetal suggested that there are tissue-specific responses of miR-210 in
tissues. both transformed and primary cell types [46]. Curiously, whilst
We previously found that the high-altitude human placenta is miR-210appearstobeamongstthemostrobustlyandconsistently
characterised by elevated levels of antioxidant molecules and a upregulatedmicro-RNAsinhypoxiainsomecarcinomacelllines
lower ATP/ADP ratio, suggesting a diminished energy reserve [24,46,47], it is downregulated in others [48]. Studies on BeWo
compared with the sea-level placenta [28]. We therefore cells [47], found an induction of miR-210 after 24h and 48h of
hypothesised that altered mitochondrial function plays a role in incubation at 1% O . Since JEG3 cells are metabolically very
2
the placental response to hypoxia and investigated the effects of active, it is possible that they induce miR-210 for a shorter time
chronic hypoxia on mitochondrial energy metabolism in human period than placental fibroblasts, and this might account for the
placentalcellsandthehigh-altitudeplacenta.Wefoundaspecific variability seen after 4 d of hypoxic exposure. In support of this,
decrease in the mitochondrial oxidative capacity of both JEG3 miR-210wasupregulatedinisolatedtrophoblastcellsafterashort
cellsandfibroblastsinhypoxiawiththeETScomplexIsubstrates, exposure to hypoxia over 8h [27]. Based on these findings, we
glutamate and malate. Conversely, respiration supported by the examined ISCU1/2 and COX10 [24] in the two placental cell
complex II substrate, succinate, was unaffected by hypoxia. types. As expected, we found a downregulation in mRNA and
Complex I protein levels were also lower in both hypoxic protein levels of both COX10 and ISCU1/2 in placental
fibroblasts and JEG3 cells; though notably transcript levels of fibroblasts. In JEG3 cells, hypoxia led to higher transcriptional
NDUF8, a subunit of complex I, were normal in all models, levelsofISCU1/2andCOX10,butsurprisinglytheproteinlevels
suggesting that a post-translational mechanism underlies this weredrasticallylowerdespitetheapparentabsenceofamiR-210
defect. Complex II protein levels were unaffected by hypoxia in induction. While these results strengthen the direct correlation
cultures, in agreement with normal respiratory rates with between miR-210 and its targets in fibroblasts, an additional
succinate. mechanism islikelytobeat playin JEG3cells.
Complex I (NADH dehydrogenase) is one of the initial Recent published data from our colleagues [28] demonstrated
complexes of the ETS, and accepts electrons from NADH to ERstressinhigh-altitudeplacentalsamples.Whenculturedunder
reduce ubiquinone. It is, however, a source of the superoxide the same conditions (1% O2) JEG3 cell proliferation rates were
anion [36], and the decreased activity we report here, which decreased, but fibroblasts were not, Reinforcing the idea of cell-
correspondswithfindingsinhypoxichumanskeletalmuscle[18], typespecificmechanisms,Yunget al.showedthathypoxiainduced
may represent a mechanism to protect mitochondria against higher levels of eIF2a phosphorylation in JEG3 cells than in
oxidativedamage.WhilstthemajormitochondrialsourceofROS fibroblasts [28].
inhypoxiaislikelytobecomplexIIIratherthancomplexI[37],a Given these observations, we assessed the effects of a protein
decreaseincomplexIactivitycouldhavetheeffectofsuppressing synthesisinhibitor(salubrinal)onthelevelsofETScomplexesand
theentryofelectronsintotheETS,decreasingROSproductionat of COX10 and ISCU1/2 in vitro. In fibroblasts, levels of both
Complex III [38]. Moreover, in hypoxic JEG3 cells, though not complexes I and IV were lower, reminiscent of the changes in
fibroblasts,wealsofounddecreasedcomplexIIIproteinlevels.In hypoxia. Interestingly, COX10 levels remained unchanged with
support of a protective, antioxidant role, we found that mtDNA onlyamildreductioninISCU1/2levelssupportingtheideathat
contentwasunchangedinbothJEG3cellsandfibroblastscultured miR-210inductionisresponsibleforthesechangesinhypoxia.In
atdifferent oxygenconcentrations.MtDNAisknowntodecrease JEG3cells,levelsofallETScomplexes,plusCOX10andISCU1/
intissuessuchasmuscleinresponsetoacuteoxidativestressand 2 were lower than controls, similar to the trend in hypoxia and
this loss is exacerbated by chronic hypoxia [39]. Moreover, the highlighting protein synthesis inhibition as the major mechanism
lack of changes in citrate synthase levels indicates an intrinsic operating in this cell type. Together, these data indicate that
response within mitochondria as opposed to a decrease in overlapping mechanisms operate in tissue-specific manner in
mitochondrial mass. hypoxia, ultimately compromising mitochondrial function, and
Wealsorecordeddecreasedrespirationrateswiththecomplex therefore cellular energetics.
IV substrates TMPD and ascorbate in both cell types, suggesting Finally, we investigated whether features of the mechanisms
impaired electron transfer to the final acceptor, O . Similarly, described in our invitro models were present in placentas from
2
protein levels of complex IV (cytochrome c oxidase) were high-altitude.Here,wefoundsignificantupregulationofmiR-210
significantly decreased in both cultures at 1% O . Curiously, inhigh-altitude placentas,whichwassimilartohypoxicplacental
2
transcript levels of mtCOX2 (a subunit of complex IV) were fibroblasts, though the increase was milder in comparison. This
unaffected byhypoxiainJEG3cells,butdecreasedinfibroblasts, maybeduetothepartialcontributionoffibroblaststothewhole
suggestingdifferentregulatorymechanisms.Adecreasedcomplex human placenta, though fibroblasts do account for the largest
IVactivitywouldservetomatchtheoxygendemandsofplacental populationofcellsinatermplacenta,oralternativelytoamilder
tissuestothediminishedsupply,suppressingoxidativemetabolism degree of hypoxia invivo compared to the 1% O culture used
2
toprotectagainstanoxia-inducedcellularinjury[40],butperhaps here. This upregulation correlated with a loss of COX10 and
also to allay the risk of fetal oxygen deprivation. Much is known ISCU1/2.Correspondingly,levelsofETScomplexsubunitswere
abouttheregulationofcomplexIVactivityinhypoxiccells,with lower in the high-altitude placenta than at sea-level, though this
hypoxia itself restricting O supply to the complex and HIF- wasnotreflectedinthemRNAlevelsofthesesubunits,suggesting
2
dependent upregulation ofinducible nitricoxidesynthase (iNOS) a post-translational repression. This may be due to miR-210
generatingnitricoxide(NO)[41].NOcompeteswithO ,thereby upregulation, or protein synthesis inhibition, which Yung etal.
2
reversibly inhibiting complex IV [42], and under sustained levels [28] found to be present in the same placentas as were studied
of NOcomplex Iisalso inhibited[43]. here.Thesuppressionofmitochondrialrespirationinthesetissues
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