Stimulation of a1a Adrenergic Receptors Induces Cellular Proliferation or Antiproliferative ...

12 Pages·2013·3.95 MB·English

Stimulation of a Adrenergic Receptors Induces Cellular
1a
Proliferation or Antiproliferative Hypertrophy
Dependent Solely on Agonist Concentration
Beilei Lei1, Debra A. Schwinn2, Daniel P. Morris3*
1DepartmentofAnesthesiology,DukeUniversityMedicalCenter,Durham,NorthCarolina,UnitedStatesofAmerica,2DepartmentsofAnesthesiology,Pharmacology,
Biochemistry,UniversityofIowaCarverCollegeofMedicine,IowaCity,Iowa,UnitedStatesofAmerica,3DepartmentofAnesthesiologyandPainMedicine,Universityof
Washington,Seattle,Washington,UnitedStatesofAmerica
Abstract
Stimulationofa AdrenergicReceptors(ARs)isknowntohaveanti-proliferativeandhypertrophiceffects;however,some
1a
studies also suggests this receptor can increase cell proliferation. Surprisingly, we find the a AR expressed in rat-1
1a
fibroblasts can produce either phenotype, depending exclusively on agonist concentration. Stimulation of the a AR by
1a
high dose phenylephrine (.1027M) induces an antiproliferative, hypertrophic response accompanied by robust and
extendedp38activation.Inhibitionofp38withSB203580preventedtheantiproliferativeresponse,whileinhibitionofErkor
Jnk had no effect. In stark contrast, stimulation of the a AR with low dose phenylephrine (,1028 M) induced an Erk-
1a
dependent increase in cellular proliferation. Agonist-induced Erk phosphorylation was preceded by rapid FGFR and EGFR
transactivation;however,onlyEGFRinhibitionblockedErkactivationandproliferation.Thegeneralmatrixmetalloprotease
inhibitor, GM6001, blocked agonist induced Erk activation within seconds, strongly suggesting EGFR activation involved
extracellular triple membrane pass signaling. Erk activation required little Ca2+ release and was blocked by PLCb or PKC
inhibitionbutnotbyintracellularCa2+chelation,suggestingCa2+independentactivationofnovelPKCisoforms.Incontrast,
Ca2+ release was essential for PI3K/Akt activation, which was acutely maximal at non-proliferative doses of agonist.
Remarkably,ourdatasuggestsEGFRtransactivationleadingtoErkinducedproliferationhasthelowestactivationthreshold
of any a AR response. The ability of a ARs to induce proliferation are discussed in light of evidence suggesting
1a 1a
antagonisticgrowth responses reflect native a AR function.
1a
Citation:LeiB,SchwinnDA,MorrisDP(2013)Stimulationofa AdrenergicReceptorsInducesCellularProliferationorAntiproliferativeHypertrophyDependent
1a
SolelyonAgonistConcentration.PLoSONE8(8):e72430.doi:10.1371/journal.pone.0072430
Editor:ClaudioM.Costa-Neto,UniversityofSa˜oPaulo,Brazil
ReceivedApril19,2013;AcceptedJuly8,2013;PublishedAugust22,2013
Copyright:(cid:1)2013Leietal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted
use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Thisworkwassupportedby2005–2016NIH/NHLBIR37HL49103(MERITAWARD):SubcellularDistribution&Regulationofa1a-AdrenoceptorstoDebra
A.Schwinn,ProjectNumber:5R37HL049103-19;$1,250,000/5years(Years16–20;MERITawardbeganinyear11).NHLBIwebsite:http://www.nhlbi.nih.gov.The
fundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction opening of the IP3R channel, while membrane bound DAG
activatesnovelproteinkinaseC(PKC)isoforms(d,e,g,mandh)
AdrenergicReceptor(AR)stimulationbyepinephrinehasbeen andincombinationwithCa2+;activatesfourtypicalPKCisoforms
recognized as integral to the fight or flight response [1] of the (a, b, b , c). DAG can also induce Ca2+ entry from the
sympatheticnervoussystemsinceearlyinthe20thcentury[2].As extracIellulIaIrmediumthroughcanonicaltransientreceptorpoten-
part of the sympathetic response, these receptors are activated tial channels [6], while depletion of ER Ca2+ stores can lead to
within seconds of stimulus recognition; however, they are also store operated Ca2+ entry through calcium release activated
involved in more extended processes including tissue injury and calciumchannels[7].Regulationofthese[8],andprobablyother
repair. Early studies distinguished the a1AR family from the [9] channels, produce the extended increase in cytosolic Ca2+
a2ARs and bARs on a pharmacological basis using specific associatedwitha1aARactivation[10–13].Inaddition,Gqappears
agonistsandinhibitors[3].EvenbeforeidentificationoftheDNA to directly activate signaling through effectors including GRK2
sequencesencodingthethreea1ARgenes,itwasrecognizedthat [14] and RhoGEFs [15] with the later activating Rho/Raf
a1ARs induced smooth muscle contraction through Ca2+ release GTPases.Althoughlimitedinformationisavailableforthea1ARs,
[4] directedby phospholipase Cbeta (PLCb). stimulation of GPCRs also activates Gbc subunits, which signal
Canonical a1aAR signaling is initiated by agonist stimulation through a variety of molecules including some isoforms of PLCb
that allows GTP association with Gq, dissociation of the trimeric [16]. In addition, Gq-coupled receptors can transactivate EGFR
GproteinsandactivationofPLCbviadirectinteractionwithGq/ and other Receptor Tyrosine Kinases through triple membrane
GTP [5]. Resultant cleavage of membrane-bound phosphatidyli- pass (TMP) signaling that involves matrix metalloproteases
nositol 4,5 bisphosphate (PIP2) produces soluble inositol triphos- cleavage of growth factor precursors [17–19]. Other signaling
phate(IP3)andmembrane-bounddiacylglycerol(DAG).Inmost proteinsreportedlyactivatedbya ARsincludePKD1[20],PLA2
1
cells,IP3inducesacutereleaseofintracellularCa2+storesthrough [21], PLD [22], AMPK [23] and Na+/H+ exchangers [24].
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a AdrenergicReceptorandCellGrowth
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Despitetheextensivestudy,mechanismsofa ARfunctionappear hydroxyphenyl)-ethylamineomethyl)-tetralone([125I]HEAT),[Per-
1
tobeverycomplexandarepoorlyunderstoodinmosttissues[25]. kin Elmer Life Sciences, Boston, MA]; High Glucose Dulbecco’s
Functionally, the a ARs are present in many cell types where Modified Eagle Medium (11995) and Pen/Strep (15140) [Gibco,
1
they play diverse roles: however, attention has focused on stress Grand Island, NY]; Hyclone Fetal Bovine Serum
responses associated with the cardiovasculature. Although a AR (SH30071.03HI),Restorestrippingbuffer(21059)andSupersignal
1
signaling can be identified by phenylephrine (PE) activation, the substrate (34076) [Thermo Scientific, Rockford,IL].
subtypethatproducesaspecificbiologicalresponsecanbedifficult
to establish in native tissues. Pharmacologic identification of the Cell Culture
a ARismoredependable,asselectiveagonistsandinhibitorsare Rat-1cellsstablyexpressinghuman,hemagglutinin(HA)tagged
1a
available for this subtype [26]. Nevertheless, transgenic mice a -AR at about 1.77pmol/mg of total protein [49] were
1A
missing individual a AR subtypes have proven invaluable, maintained in complete media containing DMEM, 10% FBS,
1
although murine phenotypes can be altered by small amounts of penicillin/streptomycin (P/S) and 400mg/ml G418. Prior to all
the remaining subtypes [27,28], as well as compensatory experiments, cultures near confluence but not quiescent were
upregulation [29] and synergistic interactions. Almost unstudied trypsinized and plated in 6 or 12 well plates and grown in
aredifferencesina ARsubtypeexpressionwithindistinct[30,31] complete media without G418 selection. For growth assays with
1
and similar [32] cell types of a single tissue, despite the potential variedPEconcentrations(i.eFig.1)cellswerewashedtwicewith
importance of endocrine like growth factor release produced by serumfree(SF)media(DMEMwithP/S)andthenreturnedtoSF
transactivation. media and immediately stimulated with the a -AR selective
1
Most studies of a AR mediated cell signaling have been agonist,PE,attheindicatedconcentrationsfor1,2or3days.For
1a
performedinexpressionmodelsusingepitopetaggedreceptorsnot Western analysis or growth assays involving pretreatment with
only because of the clarity provided by expression of a single agents,cellswerewashedtwicewithSFmediaandthenincubated
subtype, but also because native receptor levels are too low for inSFmediafor3–4hourspriortopretreatmentwithagentsand
antibodydetection[33].Inthesemodels,comparisonofsignaling stimulationwithPEfor1day.Unlessindicated,agentswereadded
efficacybetweenindividualsubtypeshasshowna ARsignalingto tocells30minPriortoPE.ForWesternanalysiscellswereplated
1a
bemorerobustinHeLa[10],rat-1fibroblast[22,34,35],HEK293 atappropriatedensityin6wellplates(100–200thousandcellsper
[34],SK-N-MC(1996theroux)andCHO[36]cells,althoughthe well), grown to near confluence (80–100%), washed twice with
relationship between canonical signaling intensity and a AR- DMEM and then incubated in DMEM for 3–4 hours prior to
1
induced phenotypic responses [36–39] remains unclear. Beyond treatments andPE stimulation asindicated.
signaling intensity,therearesubtype specific mechanismssuchas
therapidinternalization[40]andproliferativephenotypes[38]of Analysis of Cell Growth
the a AR that contrast with the slow internalization [41] and For growth assays, analysis of cell morphology (imaging), cell
1b
antiproliferative[38]phenotypesa AR.Insomenativecells,the number and protein per well were performed side-by-side. After
1a
a AR subtype displays unique signaling complexity, apparent as PE stimulation, images showing typical density were captured
1a
pharmacologically distinct basal conformations, either with high usingadigitalcamera.Cellswerecountedusingahemocytometer
prazosin affinity (a AR) or with low prazosin affinity (a AR), followingaPBSwash,trypsinzationandadditionofabout1 mlof
1a 1a(L)
sometimes observed in activity assays [26]. The low affinity DMEM.Totalproteinperwellwasalwaysquantitatedinparallel
phenotype, often designated as the a AR, appears to be wellsusingtheBCAproteinassayreagentkit(Pierce)withBSAas
1a(L)
importantinsomeprototypica ARmodelsofsmoothmusclecell a standard. Analysis was performed on 50 ml samples from PBS
1a
(SMC) contraction[42–45]. washed cells harvested in 250ml of lysis buffer (1% nonidet P-40
Thesignificanceoffibroblaststoinjuryresponsesinvolvingthe and0.5% sodium deoxycholate).
a AR[46],aswellaschronicstressincludingthosethatproduce
1a
cardiomyopathy [47,48] has increased the need to understand Western Blotting
a1aAR biology in fibroblasts. Recently, we identified a naturally Cells in DMEM for 3 to 4 hours were pretreated with agents
occurringa1ARSNPfromahypertensivepatient,whichincreases andstimulatedwithPEatthetimesandconcentrationsindicated
proliferation of rat-1 fibroblasts [49] through a mechanism prior to media aspiration and direct solubilization with 2% SDS
involvingconstitutivetransactivationofEGFR[50].Inthecurrent sample buffer. Samples were sometimes frozen in Liquid N ,
2
study, we investigated the connections between a1aAR signaling heatedat95uCfor5 min,andthensubjectto15secofsonication
pathways and biological phenotype following the discovery that or hard vortexing to shear DNA. Samples, loaded equally and
wild-type a ARs can induce either anti-proliferative or prolifer- resolvedon26well4–20%or10–20%precastCriterionSDSgels
1a
ative responses dependent solely on differences in signaling were transferred to polyvinylidene difluoride (162–0177) or
intensity due to agonist concentration. Because clonal rat-1 nitrocellulose (162–0015) membranes in standard buffers with
fibroblasts in the same media are identical prior to low or high 10%methanoland0.005%SDSusingaCriterionBlotter(plate),
dose a AR stimulation, causal and coincident signaling events all from Bio-Rad, (Hercules, CA). Western treatment conditions
1a
canbedistinguishedcompletelyfreeofsuperfluousdifferencesdue used TBS with 0.1% Tween, 5–10% dry milk (Biorad) with
toenvironment, clonalvariationandcelltype. antibodies, four 5 min, 40ml washes, and1h, 25uC,2u antibody
incubations.Primaryantibodies(targetnamed)wereincubatedat
Methods 25uC (1–3 h) or 4uC (overnight) at $ 1/1000 dilutions unless
indicated including Erk (9102), P-Erk1/2 (9101), p38 (9212), P-
Materials p38 (9211), JNK (9255), P-JNK (9251) at 1/500, Akt (9272), P-
Reagentsandsupplierswere:phenylephrine,prazosin,U73122, Akt-T308 (2965), P-Atf2 (9221) and P-Mk2 (3041) from Cell
PMA,GF109203X,genistein,thapsigargin,HRP-conjugatedgoat SignalingTechnology,(Beverly,MA)andP-FGFR3-Y724(33041)
antimouse IgG (A9169) and antirabbit IgG (A9044), [Sigma, St. at1/500fromSCBT,(SantaClaraCA)orP-EGFR1068(324867)
Louis,MO];G418,calphostinC,BAPTA/AM,W-7,KN-93,PD fromEMDMillipore.Asrequiredforthesubstrate,dilutedHRP
98059, SB 203580 (Calbiochem, San Diego, CA); 125I-(2-b-(4- secondary antibodies were used at ,1/50,000 with PVDF and
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Figure1.Thea ARinducesproliferationatlowPEconcentrationsandantiproliferativehypertrophyathighPEconcentrations.
1a
Rat-1cellsstablyexpressingthea1aARat1.77pmol/mgoftotalproteinwereincubatedat37uCunder5%CO2inserum-freeDMEMfor24,48and
72hwiththeindicatedconcentrationsofPE(10210to1025M).TheeffectofPEona AR-inducedproliferativeandhypertrophicgrowthresponses
1a
wasdeterminedbyside-by-side(A)morphologicalobservation,(B)cellcounts;(*),P,0.05at48and72h;(**),P,0.05at24h,(C)proteinquantitation
perwell;(*),P,0.05at48h;(**)P,0.05at48and72h,and(D)estimationofproteinpercell;(*),P,0.05atalltimes.Allcomparedby1-wayANOVAto
basal(n=3–8).ERepresentativeWesternblotimagesandsemi-quantitativeanalysisofhistoneH3proteinlevelsat20and24hours.
doi:10.1371/journal.pone.0072430.g001
,1/10,000 with nitrocellulose membranes. When possible mul- without native adrenergic receptors derived from embryonic
tiply probed blots were mildly stripped with Restore stripping fibroblasts[51].Intheseexperiments,rat-1cellsstablyexpressing
bufferfor5minutesat25uCorharshlystrippedwith2%SDSplus HA-a AR were incubated for 24, 48 and 72 hours with various
1a
0.7% b-mercaptoethanol at 50uC for 30 minutes. Signal was concentrationsofPE(10210to1025M)andtheeffectsofagonist
detected with X-ray film or when noted imaged and quantitated stimulation on proliferative and hypertrophic growth responses
with a HD2 CCD camera (Alpha Innotech, San Leandro, CA). determined by side-by-side cell counts, total cellular protein
Semi-quantitative analysis of band intensity from x-ray film was measurement and morphological observation. As previously
done withImageJ usingnon-saturated exposureswith membrane reported[38,39],highdosesofPE(1026to1025M)arestrongly
background. antiproliferative and induced a visually evident decrease in cell
number (Fig. 1A) quantitatively established by cell counting
Statistical Analysis (Fig.1B).Thisstrongcellcycleblockadewasaccompaniedbyan
Results are expressed as the mean6SEM, compiled from n obvioushypertrophicresponse[38],reflectedintheneardoubling
replicate experiments each performed in duplicate or triplicate. oftheproteinpercell(Fig.1Aand1D).Inthisclonalline[52],the
Statistical significance was analyzed by one-way or two-way dosedependence of thishypertrophicphenotypeon PE (Fig.1D)
ANOVA and where identified, respective, Dunnett or Bonferri was similar to the dose dependence of IP3 formation (EC50
post-tests.AllcalculationswereperformedusingGraphPadPrism ,361027M),whichservesasameasureofGproteinactivation.
(GraphPad Software, San Diego, CA) with p,0.05 considered More unexpectedly, we found low concentrations of PE
significant. (,1028M) to be associated with increased proliferation relative
to untreated control cells (Fig. 1B). Agonist dose response curves
Results alsoshowedhigherlevelsofhistoneH3incellsexposedtominimal
PE, indicative of increased DNA synthesis [53]. This unexpected
Biological Effects of a AR Stimulation growthresponsepromptedustoreconsidertheabilityofa ARto
1a 1a
Althoughthea ARshavebeenshowntoactivateawidearray regulate p38, JNK, and Erk1/2, as these MAPKs are key
1
of stress and growth related pathways, increased proliferation is regulators ofcell growth.
not a commonly observed phenotype. Thus it was initially
surprising when a AR stimulation by low doses of agonist
1a
increasedproliferationofrat-1fibroblasts;afrequentlyusedmodel
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Activation of Stress Activated Protein Kinases Activation of p38 is Required for the PE-induced
ThestressactivatedMAPKs,p38andJnk,werebothactivated Proliferative Blockade
following stimulation of HA-a1aAR expressing rat-1 cells with To investigate the mechanism of a1aAR induced, antiprolifer-
1025 MPE(Fig.2A,toppanel).Followinganacuteperiod(2 min) ative hypertrophy, we employed inhibitors of MAPKs, including
where low basal p38 phosphorylation decreases modestly, p38 thep38kinaseinhibitor,SB203580,theJNKinhibitor,SP600125,
phosphorylation became intense within 5 minutes and maximal and the MEK inhibitor, PD98059, which blocks Erk phosphor-
near 15 to 30 minutes. Thereafter p38 phosphorylation levels ylation.CellsinSFmediawerepretreatedfor30minwithvehicle
decreasedfrom1to24hours,butremainabovebasallevelsforat orinhibitorsandthenincubatedwith10 mMPEfor24hours.As
least 24 hours. Dose response experiments at several PE shownabove,PEathigherdosessignificantlyinhibitedcellgrowth
concentrations show the extent of p38 phosphorylation at 15 (Fig.3A) and increased cell size(Fig. 3B) relative tounstimulated
minutes(Fig.2B,toppanel)isalsosimilartothedosedependence control cells. As for Cho cells [36], the p38 inhibitor, SB203580,
of IP3 formation [49,52]. Compared to p38 activation, JNK prevented the antiproliferative response induced by a -AR
1a
phosphorylationisbothdelayed[54]andmoretransient,reaching stimulation in rat-1 fibroblasts (Fig. 3A); however, the inhibitor
a maximum near 30 minutes before waning rapidly (Fig. 2A, hadnosignificanteffectoncellhypertrophy(Fig.3B).NeitherErk
middle panel). The PE dose dependence of Jnk phosphorylation or Jnk inhibition interfered with either PE induced phenotype;
(Fig. 2B, middle panel) displayed a profile grossly similar to p38 however, the a AR inhibitor, prazosin, completely reverses both
1
activation. cellcycle blockadeand hypertrophy.
High doses of PE Produce Sustained Erk Inhibition Proliferation Induced by Low doses of PE Required Erk
Althougha ARstimulationcanproducemodestErkactivation Activation
1a
[36], in rat-1 cells high doses of PE (1025M) inhibit Erk activity Given the established importance of MAPKs in control of cell
(Fig. 2A, lower panel) in agreement with prior evidence [37]. growth [55], the role of MAPKs and upstream activators in low
Although Erk phosphorylation was severely reduced both acutely dosea -ARsignalingwasinvestigatedunderconditionsotherwise
1a
(2 min) and later in the time course (,1 hour), we did observe a identical to those used in the high dose experiments. Inhibitor
period of recovery between 15 and 30 minutes (Fig. 2A, lower analysis showed that p38 and Jnk signaling were not involved;
panel) where Erk phosphorylation approached basal levels. At however, inhibition of Erk signaling largely blocked proliferation
even longer times, Erk phosphorylation remained depressed induced by 1028M PE (Fig. 4A) at concentrations of the MEK
relative to untreated control cells. Problematically, the basal Erk inhibitor, PD98059, that also prevented increased Erk phosphor-
phosphorylationlevelofcellsplacedintoSFmediafor3–4hours ylation (Fig. 4B). Consistent with control of proliferation, the
was both low and sensitive to minor details of cell handling, additionof1028MPEincreased ERKphosphorylationwithin2
nevertheless,theresponsepatternincludingbothminimaandthe minutesandproducedstrongactivationbetween5and30minutes
recovery period was qualitatively consistent (n.13). A dose (Fig. 4C). In contrast, P-Jnk was undetectable under basal and
response curve directed at the time of acute Erk dephosphoryla- stimulatedconditions(datanotshown).Stimulationwith1028M
tion (,2min), defined a broad range of agonist concentrations PE produced p38 phosphorylation that was marginally (e.g.
(1027 to 1025M PE) that produced this acute inhibitory effect Fig. 2B) but not significantly higher than detectable basal levels
(Fig.2Blowerpanel).Inaddition,thiscurveshowedanincreasein (1.2960.15-fold,n=6)andneverexceeded1.8-foldoverbasalin
Erk phosphorylation at 1028M PE relative to basal levels, anylow doseexperiment.
suggestinganarrowrangeofconcentrationswithinwhicha -AR
1a
stimulationcanincreaseErkactivity.Giventheestablishedroleof EGFR Transactivation Precedes and is Required for Low
Erk in enabling proliferation, the elevated phosphorylation dose PE Activation of Erk
associated with low doseagonist stimulation suggested a basisfor
Becausetransactivationofreceptortyrosinekinases(RTKs)was
increased proliferation (Fig.1B) that isaddressed below.
a potential mechanism of Erk activation [17], we analyzed the
activation-dependent phosphorylation state of the fibroblast and
general growth receptors, FGFR and EGFR. Following a -AR
1a
stimulation, phosphorylation of both RTKs exhibited temporal
Figure2.Westernanalysisofhighdosea ARinducedMAPKactivationusingphospho-specificantibodiesforactivatedkinases.
1a
a ARexpressingcellswereplacedintoSFmediafor3–4hours,treatedwithPEfortheindicatedtimeandcollectedbydirectadditionofSDSsample
1a
buffer.(A)Timecoursefollowingtreatmentwith1025MPEshowsstrongp38activation,transientJnkactivationandacuteinhibitionofalreadylow
basalERKactivityfollowedbytransientpartialrecovery.(B)DoseresponseofPEandMAPKphosphorylation.Followingtransfertoserumfreemedia
thecellsweretreatedfor15or2minwith0to1025MPEasindicated.
doi:10.1371/journal.pone.0072430.g002
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Figure3.TheantiproliferativeeffectsofhighdosePErequire
p38 activity. a AR expressing cells placed into SF media were
1a
pretreatedfor30minwithvehicle(0.1%DMSO),20mMPD98059(MEK/
Erk inhibitor), 10mM SB203580 (p38 inhibitor), 10mM SP600125 (Jnk
inhibitor),or10mMPrazosin(a1AR)andthenstimulatedwith10mMPE Figure 4. Low dose a AR stimulation activates growth-
for24hpriorto(A)cellcountingor(B)proteinassayandestimationof 1a
associated signaling. a AR expressing cells were placed into SF
cellularproteincontent.Valuesarethemean6SEM(n$3);(*),P,0.05, 1a
mediapriortopretreatmentandstimulation.(A)Priortocounting,cells
comparedtovehicleorinhibitoralone.
werepretreatedfor30minwithvehicle(0.1%DMSO),20mMPD98059
doi:10.1371/journal.pone.0072430.g003
(MEK/Erk inhibitor), 10mM SB203580 (p38 inhibitor), 10mM SP600125
(Jnk inhibitor), or 10mM Prazosin (a AR) and then stimulated with
1
patterns that were recognizably similar to one another (Fig. 4C, 10mM PE for 24h. Values are the mean6SEM (n$3); (*), P,0.05,
upper panel), despite P-EGFR signal near the limit of detection. comparedtovehicleorinhibitoralone.(B)20mMPD98059blocksPE-
inducedErkphosphorylation.(C)Erkisacutelyactivatedwithin2minof
RapidbuttransientphosphorylationoftheRTKeffector,Akt,was
10mM PE addition and remains robustly phosphorylated for 15–30
alsoobservedfollowingRTKphosphorylationbutwastemporally minutes. Prior to Erk activation, stimulation induces sustained
distinctfromErkactivationandwaslostasErkreachedmaximal phosphorylationofEGFRandFGFRandacutetransientphosphorylation
activity. Semi-quantitative estimation of relative band intensity ofAktatT308downstreamofPI3K/Aktsignaling.TotalAktprovidesthe
(Fig.4C,lowerpanel)illustratesthetemporaldistinctionsbetween load control for this multiply reprobed blot. In the lower panel,
differencesintemporalactivationpatternsareshownfollowingsemi-
these activation profiles.
quantitative analysis of Erk, FGFR and Akt phosphorylation. Results
ConsistentwithRTKinvolvement,thegeneralRTKinhibitor,
representthefoldincreaseinphosphorylationrelativetobasalsignal
genistein, completely prevented the a1aAR induced proliferative (Erk,FGFR3)orforAkt,background(arbitrary)signal.
response (Fig. 5A) as well as increased Erk phosphorylation doi:10.1371/journal.pone.0072430.g004
(Fig.5B).Toidentifythepathwayresponsible,specificinhibitorsof
potential RTKs were tested. While the FGFR inhibitor,
To demonstrate that TMP signaling was the mechanism of
PD173074, modestly decreased proliferation (Fig. 5A) and Erk
EGFR transactivation responsible for increased Erk activity,
phosphorylation(Fig.5B)ofbothbasalandstimulatedcells,itdid
various concentrations of the commonly employed ‘‘general’’
notappeartoblocktheincreaseinproliferationandErkactivation
MMP domain protease inhibitor, GM6001 (galardin), were
induced by low doses of PE. In contrast, the EGFR inhibitor,
applied to cells 30 minutes prior to PE stimulation (Fig. 5D).
AG1478, had little impact on either basal proliferation or Erk
Across the PE concentrations that induce proliferation, GM6001
phosphorylation, but largely prevented both agonist-induced
reduced receptor-activated Erk phosphorylation in a dose depen-
proliferation and Erk activation (Fig. 5A and 5B). Inclusion of
dent manner. As TMP signaling is predicated on growth factor
bothinhibitorsresultedinapparentlyadditiveeffectsoflowbasal
precursor proteolysis occurring outside the cell, competitive
proliferation and minimal agonist-induced proliferation. Because
inhibition by GM6001 [58] should be almost instantaneous, as
preliminary experiments showed 100mM concentrations of
confirmed by the similar results obtained with a GM6001
AG1478 and a second EGFR inhibitor, Erlotinib, completely
preincubation of ,15 seconds (Fig. 5D, lowest panel). These
blockedErkactivationevenwithashort5minutepreincubation,
results strongly suggest TMP transactivation is essential for low
dose response experiments were performed which demonstrated
respective IC50s 865 nM and 3064 nM for these inhibitors dose a1aAR activation of Erk and increased proliferative of rat-1
cells.
(Fig. 5C). These results are consistent with the potent EGFR
inhibitionpreviouslyreported[56,57]andsuggestintendedtarget
inhibition.
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a AdrenergicReceptorandCellGrowth
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Figure5.EGFRtransactivationthroughaTMPmechanismisrequiredforlowdosea AR-inducedproliferationandErkactivation.
1a
a ARexpressingcellswereplacedintoSFmediapriortopretreamentandadditionofPE.(A)Priortocounting,cellswerepretreatedfor30minwith
1a
0.1%DMSO(Veh),20mMGenestein,1mMPD173704(PD17),1mMAG1478(AG),1mMPD173704+1mMAG1478priortogrowthwithout(control)or
with 1028M PE.Values arethe mean6SEM;(*), P,0.05comparedto vehicleor inhibitoralone(n$3);(**), P,0.05comparedtovehicle+PE.(B)
RepresentativeWesternanalysisofErkphosphorylationincellspretreatedfor30minwithvehicle0.1%DMSO(Veh),1mMPD17,1mMAG,1mMPD
+1mMAGor20mMgenistein(Gen)priortostimulationwithout(2)orwith(+)PEat261028Mfor10minutes.Lowerpanelshowssemi-quantitative
analysisofP-ErkbandintensityfromCCDimages.Valuesarethemean6SEM(n=4);(*),P,0.05comparedtovehicleorinhibitoralone.(C)Inhibition
ofErkphosphorylationbyadilutionseriesoftheEGFRinhibitors,AG1478andErlotinib,added5minutespriortostimulationwith361028MPEfor5
minutes.Tomaintainrelativeintensity,panelswereconcordantlyadjusted.(D)QuantitationofP-ErkbandintensityattheindicatedAG1478and
Erlotinibconcentrationsrelativetolevelsinuntreatedcells.(E)Top3panelsshowswesternanalysisofErkphosphorylationincellspretreatedfor
N
30minwith0.1%DMSO(Veh)orGM6001(GM)priortoincubationfor5minuteswithout(2)orwith(+)PE.Thebottompanelshowsasimilar
experimentinwhichtheGM6001preincubationwas,15seconds.Paneladjustedindependentlytoemphasisinhibitionpattern.( )Indicatesan
artifactualband.
doi:10.1371/journal.pone.0072430.g005
Erk Activation RequiresPLCbProduction ofDAG but not ing IP3R-mediated Ca2+ responses through a mechanism inde-
Increased Intracellular Calcium pendent of Gq/PLCb signaling. However, these authors found
Gq/PLCb/IP3Riscompletelyinhibitedby10 mMU73122inless
Although the ability of Gq-coupled GPCRs to transactivate
than3minuteswhereasthelargerERCa2+transientsmediatedby
EGFRleadingtoErkmediatedproliferationiswellestablishedin
caffeine-activated ryanodine receptors was impacted more slowly
rat-1 cells [17,59–61]], the role of canonical Gq signaling in this
(.4 min), suggesting maintenance of adequate ER Ca2+ levels
process has not been investigated. Consistent with a requirement
for Gq activation, inhibition of PLCb with U73122, effectively across this period. Using a short, 3 minute, preincubation, we
found the initial PE-induced increases in Erk phosphorylation
blocked increased Erk phosphorylation (Fig.6A). In these images
were inhibited by 2 mM and blocked by 5 mM U73122 (Fig. 6A,
somebandshavebeenoverexposedtoallowvisualizationofbasal
2 min PE). Note that basal Erk phosphorylation at that time
Erk activity; however, these long exposures show the variable
(3 min preincubation plus 2 min incubation) was unaffected by
phosphorylation of the p44 Erk isoform, providing an accurate
5 mMU73122,suggestingminimalimpactonErksignalingatthe
proxyforp42phosphorylationinlessexposedimages.Recently,it
time of receptor stimulation (3min). Erk phosphorylation follow-
has been reported that U73122 can also inhibit the SERCA
ing5minuteofPEstimulationwassubstantiallyreducedby5 mM
calciumpump[62],potentiallyemptyingERstoresandsuppress-
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a AdrenergicReceptorandCellGrowth
1a
Figure 6. Low-dose a AR-induced proliferation requires Erk phosphorylation dependent upon Gq signaling through PLCb and
1a
PKC.a ARexpressingcellswereplacedinSFmediapriortopretreatmentandstimulation.(A)WesternanalysisofcellspreincubatedwithDMSOat
1a
0.1%(V2)or0.25%(V5)orwiththePLCbinhibitor,U73122,priortostimulationwithout(2)orwith(+)361028MPEfortheindicatedtimes.Panels
adjusted independently. (B) Western analysis of cells preincubated 10min with 0.2% DMSO (Veh) or with 40mM BAPTA-AM (B-AM) prior to PE
stimulationatthetimesandconcentrationsindicated.(C)Westernanalysisofcellspreincubatedfor15or30minuteswithwith0.2%DMSO(Veh)or
thePKCinhibitor,GF109203X,priortostimulationwithout(2)orwith(+)361028MPEfortheindicatedtimes.
doi:10.1371/journal.pone.0072430.g006
U73122(notep44isoform);however,completeinhibitionrequired inhibits basal and stimulated Akt activity (Fig. 7D). Although
a concentration of10mM(Fig.6A, rightpanels). chronic inhibition of Akt probably plays a role in enforcing the
To delineate the PLCb signaling pathway responsible for antiproliferative phenotype, these delayed effects are beyond the
transactivation we investigated dominant downstream effector scope of the current report as they are not associated with the
pathways. In contrast to PLCb inhibition, complete chelation of proliferative response and probably involve distinct changes in
intracellular Ca2+ with 40mM BAPTA-AM [63] increased basal geneexpression.
Erk phosphorylation and did not prevent acute a AR-induced
1a
Erk phosphorylation (Fig. 6B), strongly suggesting increased Discussion
cytosolic Ca2+ is not necessary for Erk activation as well as
Generally, a AR stimulation has been associated with
limiting potential problems associated with off-target SERCA 1a
antiproliferative [38] and [39,65] hypertrophic phenotypes;
inhibitionbyU73122(above).Ontheotherhand,broadspectrum
however, this receptors ability to induce proliferative [46] and
inhibition of PKC isoforms with GF109203X resulted in
protective [66] responses demonstrates a diversity of biological
concentration dependent reduction in Erk phosphorylation that
functions. This variability is consistent with contradictory a AR
wascompleteattheconcentrationof10 mMwhethera ARwas 1a
stimulated with 361028M (Fig. 6C) or 1028M PE 1(adata not signalingresponsesthatarenoteasilydistinguishedfromresponses
of the other subtypes. Consistent with phenotypic results in
shown). These results strongly suggest that Erk activation by low
expression models [38,39], pharmacologically dissection in
doses ofPErequires canonical Gqsignaling andisdependent on
primary foreskin fibroblasts suggests the a AR mediates prolif-
PLCbactivationpresumablyofnovelPKCisoforms,whichdonot 1b
eration rather than the equally expressed a AR. [67]. Indeed,
require calcium. 1a
until very recently [50], no study had reported a AR-induced
1a
EGFR transactivation, even though rat-1 cells were used in the
a AR Induced Activation of PI3K/Akt is Separable from
1a originaldissectionofTMPsignaling[17,59,60,68]andhavebeen
Erk Activation and Proliferation a primary model of a AR signaling. Nevertheless, under some
1a
EGFR signaling through Akt can be proliferative; however, conditionsthenativea ARappearstoenhanceproliferation[46]
1a
a1aARactivationofAktwastemporallycomplexandnotmaximal asweobservedwithminimalstimulationinrat-1fibroblastsdueto
at PE concentrations inducing proliferation. At low PE concen- a mechanisminvolving EGFRtransactivation.
trations, modest, acute Akt activation returned to baseline by 15 Thea ARhasbeenviewedasastressreceptorduelargely to
1a
minutes (Fig.4C, and7A); however, at higherPE concentrations agonist-induced activation of stress pathways, including sustained
more intense Akt activation is preserved for a longer period increasesincytosolicCa2+[10–13]andactivationofbothp38and
(Fig. 7A). These findings do not contradict an earlier report that Jnk[36,37,69,70],allofwhichareassociatedwithcelldeath[71]
a ARactivationinrat-1cellsinhibitsAktsignaling[64],ashigh [72]. Also induced are potentially deleterious immunologic
1a
doses of PE invariably reduced Akt phosphorylation within one pathways including arachidonic acid release [73], NF-kB activa-
hour (Fig. 7B). Indeed, chronic PE administration for 24 hours tion, IL6 secretion [74] and TNFa secretion [75] due to TACE
stronglyinhibitsAktactivityatallPEconcentrationsabovethose transactivation [76] all concordant with a AR function during
1a
associated with cell proliferation (Fig. 7C). Of equal importance wound healing responses [46,75,77]. More broadly, Gq/PLCb
and in stark contrast with Erk activation, Akt phosphorylation activationappearstohaveplayedaconservedroleinwoundingas
appears strongly dependent on intracellular Ca2+as BAPTA-AM farback asC.elegans [78].
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a AdrenergicReceptorandCellGrowth
1a
Figure7.Stimulationofa ARresultsinstrongCa2+dependentAktactivationfollowedbychronicAktdownregulationathighPE
1a
concentrations.a ARexpressingcellswereplacedintoSFmediapriortopretreatmentandstimulationwithPE.RepresentativeWesternAnalysis
1a
showingthePEdosedependenceof(A)acuteAktphosphorylationfrom2–10min.,(B)intermediateAktandErkphosphorylationat1hourand(C)
chronicAktdephosphorylationat24hours.(D)Treatmentofcellswith0.1%DMSOvehical(2)or20mMBAPTA-AM(+)for10min.priorto5min
stimulationwithvariousconcentrationofPEdemonstrateanimportantroleforintracellularCa2+releaseinAktactivationthatcontrastswithErk
behavior.
doi:10.1371/journal.pone.0072430.g007
Asastressactivatedkinase,withanestablishedroleinblocking release that required both PKC and Src activities [86]; however,
the cell cycle at both the G1/S and G2/M transitions [79], it is thesecellsreportedlyexpressmoreproliferationassociateda AR
1b
unsurprisingthattherobustandextendedp38activationinduced [21]. Although SMCs lose a ARs during isolation [30], an
1a
by a AR stimulation produces a nearly complete cell cycle elegant series ofin vivoand vessel studies bythe Faberlaboratory
1a
blockadethatcanbepreventedbyp38inhibition.Consistentwith suggest the a AR can increase proliferation of both SMCs and
1a
indirect blockade of the G1/S transition through Erk inhibition fibroblasts during vessel injury [46,77,87]. Despite the predomi-
[79], p38 phosphorylation is also associated with a reciprocal nance of the a AR in the SMCs of conduction vessels and the
1d
decrease in Erk phosphorylation. At longer times, cell cycle presenceoftheproliferativea AR[31],pharmacologicdissection
1b
blockadeisprobablyreinforcedbygeneticreprogrammingashas showsthea ARisessentialforproliferation[77,87].Ofnote,the
1a
been suggested [38,39], presumably through mechanisms related proliferativeeffectofthea ARonmedialSMCsoccurreddespite
1a
inparttoactivationofp38anditseffectors.Despiteconsiderable the near absence of this receptor from this cell type [31],
study,themechanismbywhicha ARsandotherGPCRsactivate potentiallysuggestingcelltocellendocrine-likesignalingasaresult
1a
p38 to produce an antiproliferative phenotype has yet to be oftransactivationofthefibroblastpopulation.Thea ARcanalso
1a
established. Both our laboratory [52] and others [54] have be protective as in heart [66], where activation of EGFR by Gq-
observednorequirementforcanonicalPLCbsignalingina AR- coupledGPCRsincludingthea AR/a ARmayinvolveEGFR
1a 1a 1b
mediated p38 activation. Fairly recently it has become apparent transactivationandErksignaling[88,89].Incardiomyocytes,Gq-
that both Gq and Gbc can induce RhoGEF activation of Rho coupled GPCRs activate Erk much more than PI3K/Akt [90],
GTPases [15,80,81] and that one pathway is mediated by direct potentiallysuggestingamechanismdistinctfromtransactivationin
RhoGEF association with G [82] or G [15] bypassing which growth factor release might be expected to activate both
q/11 12/13
PLCb. A number of studies have linked Rho GTPases to p38 pathways [89]. In this regard, it is notable that a ARs in rat-1
1a
activation, including a recent mechanistic description of p38 cells activate Erk at a lower concentration of agonist using fewer
activation following a AR stimulation through the RhoGEF, activated receptors, without a requirement forCa2+release.
1b
AKAP-lbc [83]. Ironically, these investigators used a ARs The role of canonical Gq-coupled signaling in EGFR
1b
expressed in HEK293 cells, where the a AR has an uncharac- transactivation has received relatively little attention and had not
1b
teristic antiproliferative, hypertrophic phenotype [84], perhaps been studied in relation to a AR signaling. More surprisingly,
1a
because transactivation is poorly coupled in these cells [85]. The giventheextensivestudyofTMPtransactivation,neitherhasthe
relevance of this pathway and other Rho/Gef signaling to p38 roleofcanonicalGqsignalingbeenaddressedinrat-1cellsforany
activationbythea ARandotherGPCRsrequiresfurtherstudy. receptor. The results reported here suggest a requirement for
1a
Giventheusualantiproliferativeandhypertrophicphenotypes, PLCb activation during EGFR transactivation by the a AR,
1a
the ability of the a AR to induce proliferation of rat-1 cells despiteminimalIP3production[52]orcalciumrelease[12]atthe
1a
through EGFR transactivation provides important support for agonist concentrations that lead to Erk activation. In addition,
invivo evidence of this phenotype. Extensive studies in rat-1 cells cytosolic chelation of this minimal Ca2+ release does not prevent
have shown most Gq-coupled receptors [ET , LPA, Thrombin Erk phosphorylation. Combined with a requirement for PKC
A
[59], M1-acetylcholine [17], BB2-bombesin, Bradykinin [60], activity,theseresultssuggesttheessentialfunctionofPLCbduring
CASR-calcium [61]] can transactivate EGFR through a TMP a AR induced proliferation is DAG activation of a nonclassical
1a
mechanism involving Hb-EGF. However, until our recent report PKC isoform.
[50] and the data presented above, the a AR was a notable Even for other Gq-coupled receptors, evidence for or against
1a
exception. An extensive analysis in GT1-7 neuronal cells had canonical signaling in transactivation is limited and disparate
suggested EGFR transactivation by the a ARinvolved Hb-EGF [19,91,92], however, some studies report PKC involvement
1a
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a AdrenergicReceptorandCellGrowth
1a
upstream of EGFR activation [19,85,93]. Problematically, many tion due to acutely released growth factors from the proliferative
studies have focused on the AT1R, which along with the a b1 effectsofEGFR/Erkmediatedgeneexpressionseemedunlikelyto
1b,
and b2 adrenergic receptors, displays rapid b-arrestin mediated bedefinitive andhas notbeen pursued.
internalization [94] that often leads to sustained cytosolic Erk TransactivationdependentsignalingbyPI3K/Aktdownstream
activationthroughintracellularbArrsignaling[94,95].Incontrast, of a ARs [26] and other Gq-coupled GPCRs [18] appears
1
the a1aAR internalizes very slowly [41,96,97] through a mecha- important for contraction of smooth muscle cells. In mesenteric
nism largely independent not only of the carboxy terminus [96] resistancearteries,wherethea AR(ora AR)isdominantatthe
1a 1L
but also receptor activation [98,99] and phosphorylation [100]. mRNA [43,104] and functional [42,105] levels, inhibition of
These characteristics suggest internalization-dependent mecha- EGFR has little impact on acute a AR-mediated contraction
1
nisms of Erk activation will be less important to a1aAR signaling [106] induced by Ca2+/Calmodulin activation of myosin light
perhaps favoringEGFRtransactivation, particularly inrat-1 cells chain kinase [107], but largely prevented sustained vessel
where signaling by focal adhesion complexes is limited [101]. contractioninducedbyPI3K/AktdownstreamofEGFRtransac-
More broadly, activation of PKC by DAG or phorbal esters is tivation [106,108]. In the current study, the role of PI3K/Akt
generally proliferative and has been implicated in transactivation signalinginproliferationwaslessclearandmaximalactivationof
[102]. Given the number of Gq-coupled receptors that can Aktbythea ARdidnotcorrelatewiththelowdoseproliferative
1a
transactivateEGFR,itseemslikelythatDAGfrequentlyfunctions response. For this reason it was not a focus of the current study,
as an initiatorof PKC induced proliferation. nevertheless, it is noteworthy that the PI3K/Akt pathway often
The proliferative response induced by minimal a1aAR stimu- supports Erk signaling at low levels of EGFR activation [109]
lationoccurredoveranarrowandsomewhatvariablerangeofPE giventhata AR-inducedtransactivationofErkwasprecededby
concentrations (1028 to 361028M) at the lower edge of the modest, brie1faAktactivation.
efficacious concentrations. Consequently, 1028M PE was some- On the other hand, robust acute Akt activation at higher PE
times ineffective, leading to frequent use of the slightly higher concentrations correlated with proliferative inhibition. However,
concentration. Nevertheless, all PE concentrations producing a
this anti-proliferative effect is more reasonably linked to chronic
proliferative response were considerably below the EC of IP3
50 Akt inhibition subsequent to p38 activation. The need for Akt
formation(PE,361027M),clearlydemonstratingatinyfraction
signaling during cellular growth suggests retention of Akt activity
ofavailablereceptorsareresponsibleforthephenotype.Although
with low agonist plays at least a permissive role in allowing
distinct fractional populations of a ARs have been identified
1a proliferation. The divergent requirement for releaseof intracellu-
[45,52,103],a1aARsstablyexpressedinrat-1cellsat,1.8pmole/ lar Ca2+ for Erk and Akt activation was unexpected and clearly
mg display unambiguous receptor reserve behavior including
suggests distinct separable signaling pathways. Of potential
agonistbindingaffinities(Kd,1025to361025M)thatare30-to
significance to fibroblasts, a AR stimulation also resulted in
100-foldabovetheEC forIP3production[49,52].Inaddition, 1a
50 transactivation of an FGFR, perhaps FGFR3, which functions
receptorsat5-foldlowerdensitydisplayabout5-foldhigherEC
50 primarilythroughPI3K/Aktsignaling[110,111].Importantly,an
values [49], implying conservation of activated receptor number.
inhibitor with specificity toward FGFR1/3 slightly reduced both
Although receptor reserve does not disprove the existence of a
Erk phosphorylation and cell proliferation without apparently
subpopulation of a ARs with special characteristics and high
1a impacting agonist-induced effects. While EGFR clearly plays an
agonistaffinity,itallowsthepossibilitythatfractionalactivationof
essential role in a AR agonist induced Erk activation and
‘‘typical’’ receptors could produce the low dose response. In any 1a
proliferation of rat-1 fibroblasts, the biological function of PI3K/
case, PLCb activated by low doses of PE drives very strong Erk
Akt signalingrequires additionalstudy.
activation despite almost undetectable increases in IP3 and
Thecombinatorialbasisofstresssignalinghasbeenrecognized
presumably DAG at the whole cell level. This finding is of
for more than a decade in well studied models of cardiac injury
considerable significance, as it suggests Erk activation by EGFR
[112]. Concordantly, we view the question of which pathways
maybethepathwaymosteasilyactivatedbyagoniststimulationof
representnativea ARsignalingasaredherring,giventhearray
a ARinfibroblasts. 1a
1a of receptors activated with the a AR during severe stress and
Somewhatdivergently,ourrecentfindingthatamutanta AR 1a
1a tissue injury. In those situations where isolated a AR signaling
(G247R) induces proliferation through constitutive EGFR trans- 1a
operatesaspartofnormaltissuefunction[e.g.duringpenilevessel
activationsuggestedamechanismthatwasGproteinindependent
contraction [113]], transactivation of Akt may represent a
[50]. While G247R-a ARs may in fact bypass the requirement
1a dominant signaling process. However, during severe stress,
for PLCb/PKC, our present data shows that very small, agonist-
combinatorial signaling can induce extreme responses, such as
induced increases in IP3/DAG can induce transactivation. This
the sustained Ca2+ elevation and p38 activation of cardiac
readilyexplainshowbasalPLCbactivityassociatedwithG247R-
ischemia [114,115] that are similar to high dose a AR response
a ARwasnotdetected,butcannotexplainwhyprazosindidnot 1a
1a in the rat-1 model. Less extensive vessel injury will be associated
significantly prevent G247R-a AR induced proliferation [50].
1a
withlessadrenergicstimulationandlowerreceptoractivationthat
One possibility is that G247R-a AR, which constitutively
1a
maysupportproliferationandvesselrepair[77,87].Indeed,recent
activates EGFR, induces enough constitute PLCb activity and
evidencethatchronicstressresponsessuchascardiachypertrophy
DAG production toenabletransactivation evenwhenprazosin is
aremediatedbyfibroblastactivation[47],suggestadditionalroles
bound. Alternatively, chronic transactivation by the mutant
for a ARs in fibroblasts [48]. While the biological reason for
receptor may have reprogrammed gene expression using well 1a
proliferative and antiproliferative signaling through the same
established EGFR/Erk based mechanisms [92], resulting in a
reduced requirement for PLCb/PKC signaling. Potential diver- receptorremainstobedetermined, theunanticipated isolationof
gence between acute and chronic signaling is also relevant to Erk signaling at the lowest agonist concentrations allowed
proliferation due to agonist-induced Erk activation; however, in unambiguous analysis of this pathway independent of concurrent
thestimulatedmodelacutelyreleasedgrowthfactorsremaininthe signaling through unrelated pathways or other a1AR family
media as evidenced by the continued elevation of FGFR members.
phosphorylation. Delineating the importance of chronic stimula-
PLOSONE | www.plosone.org 9 August2013 | Volume 8 | Issue 8 | e72430
a AdrenergicReceptorandCellGrowth
1a
Acknowledgments Author Contributions
WethankAnushOganesianforscientificconsultationandMarinKleine- Conceived and designed the experiments: BL DM. Performed the
Brueggeneyforcriticaleditingofthemanuscript. experiments: BL DM. Analyzed the data: BL DS DM. Contributed
reagents/materials/analysistools:DSDM.Wrotethepaper:BLDSDM.
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PLOSONE | www.plosone.org 10 August2013 | Volume 8 | Issue 8 | e72430