Krox20 stimulates adipogenesis via C/EBPβ-dependent and

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A R T I C L E
β
Krox20 stimulates adipogenesis via C/EBP -dependent
and -independent mechanisms
Zhu Chen,1,2 Javier I. Torrens,1 Ashim Anand,3 Bruce M. Spiegelman,3 and Jeffrey M. Friedman1,2,*
1LaboratoryofMolecularGenetics
2HowardHughesMedicalInstitute,RockefellerUniversity,1230YorkAvenue,NewYork,NewYork10021
3Dana-FarberCancerInstituteandtheDepartmentofCellBiology,HarvardMedicalSchool,44BinneyStreet,Boston,Massachusetts02115
*Correspondence:[email protected]
Summary
Krox20isazincfinger-containingtranscriptionfactorthatisabundantlyexpressedinadiposetissue.However,itsrolein
fatcelldifferentiationhasnotbeenestablished.Incultured3T3-L1cells,Krox20israpidlyinducedbyserumstimulation.
Overexpression of Krox20 in both 3T3-L1 preadipocytes and multipotent NIH3T3 cells promotes adipogenesis in a hor-
mone-dependent manner. Conversely, RNAi-mediated loss of Krox20 function reduced adipogenesis in 3T3-L1 cells. Ec-
topicexpressionofKrox20cantransactivatetheC/EBPβpromoterandincreaseC/EBPβgeneexpressionin3T3-L1preadi-
pocytes.RNAi-mediatedknockdownofC/EPBβdiminishedKrox20’sproadipogeniceffect.Finally,coexpressionofKrox20
and C/EBPβ in naive NIH3T3 cells resulted in the pronounced induction of a fully differentiated adipocyte phenotype, an
effectpreviouslyobservedonlywithPPARγ.ThesedataindicatethatKrox20isnecessaryforadipogenesisandthat,when
overexpressed,Krox20potentlystimulatesadipogenesisviaC/EBPβ-dependentand-independentmechanisms.
Introduction actively expressed and then begins to diminish around day 2
of hormonal induction, at which point the RNAs for C/EBPα
Obesity,theexcessdepositionofadiposetissue,isoneofthe andPPARγincrease(Caoetal.,1991).C/EBPαandPPARγin-
mostpressinghealthproblemsintheWesternanddeveloping duceprogramsofgeneexpressionleadingtothedifferentiation
world.Thus,themoleculareventsunderlyingthedetermination ofmatureadipocytes(Rosenet al.,2002;LinandLane,1994;
anddifferentiationofadiposetissueareofbasicandpotential Tontonoz et al., 1994). Loss-of-function genetic studies indi-
clinical importance. The immortalized mouse adipogenic 3T3- catethatPPARγisabsolutelyrequiredfordifferentiation(Rosen
L1 and 3T3-F442A cell lines have been used extensively to etal.,1999);whileC/EBPαisnotstrictlyrequiredforadipogen-
study these processes. When exposed to a cocktail of hor- esis,itisrequiredforinsulinaction(Rosenetal.,2002).
mones, 3T3-L1 cells change their appearance from that of fi- Recent studies have shown that a large number of other
broblasts to that of adipocytes. In these cells, the process of transcriptionfactorsareexpressedduringadipogenesisinvitro
adipocyte conversion follows a well-orchestrated program in andinadiposetissueinvivo(Soukasetal.,2001).Thesedata
whichtheCCAAT/enhancerbindingproteinsC/EBPδ,C/EBPβ, have suggested that a set of complex transcriptional hierar-
and C/EBPα (Cao et al., 1991; Umek et al., 1991; Yeh et al., chies and networks are responsible for the changes in cell
1995)andthenuclearhormonereceptorperoxisomeprolifera- morphology and gene expression associated with adipocyte
tor-activated receptor γ (PPARγ) (Rosen et al., 2002; Tontonoz differentiation. This network of transcription factors forms a
et al., 1994) are sequentially activated and play pivotal roles. crossregulatorycircuitwherebythedifferentiatedstatecannot
C/EBPβ and C/EBPδ are among the first transcription factors onlybeinitiatedbutalsomaintained.
induced following exposure of preadipocytes to hormonal in- Inthis report,we setoutto identifyadditionalfactors capa-
ducers, and they are both stimulatory to adipogenesis (Yeh et ble of influencing adipocyte differentiation. We reasoned that
al., 1995). Loss-of-function studies have shown that C/EBPβ suchfactorswouldbeenrichedinadiposetissueinvivowhere
andC/EBPδhaveasynergisticandessentialroleinadipocyte adipogenesis normally takes place. In earlier studies of the
differentiation(Tanakaetal.,1997). transcriptional programs associated with adipocyte develop-
In contrast, naive 3T3 cells do not readily differentiate into mentinvitroandinvivo,weidentifiedapaneloftranscription
adipocytes when exposed to the same cocktail of hormones factorsthatareexpressedatamuchhigherlevelinwhiteadi-
that lead to the differentiation of 3T3-L1 cells. PPARγ is the posetissuesinvivo,ascomparedtomatureadipocytesdevel-
only factor capable of fully differentiating naive 3T3 cells into opedinvitrofrom3T3-L1preadipocytes(Soukasetal.,2001).
adipocytes. Ectopic expression of C/EBPβ can also convert One of these genes, Krox20 transcription factor, was ex-
multipotentNIH3T3cellsintomatureadipocytes,butthiseffect pressed at high levels in adipose tissue in vivo and at much
is much less robust than PPARγ, while C/EBPδ had no effect lowerlevelsduringadipogenesisinvitro.Krox20wasoriginally
ontheadipocytedifferentiationofthesecells(Wuetal.,1995; isolated as an immediate-early response gene from serum-
Yehetal.,1995). stimulated NIH3T3 fibroblasts (Chavrier et al., 1988). Other
C/EBPβ can also promote the fat differentiation of 3T3-L1 studies showed that Krox20 is expressed in developing em-
cells.Afterexposuretoahormonalcocktail,C/EBPβmRNAis bryos and that it is tightly controlled temporally and spatially
CELLMETABOLISM:FEBRUARY2005·VOL.1·COPYRIGHT©2005ELSEVIERINC. DOI10.1016/j.cmet.2004.12.009 93
A R T I C L E
(Wilkinsonetal.,1989)inanumberofsitesincludingtheseg- son. In this and all subsequent experiments, at least 30% or
mented hindbrain (Swiatek and Gridley, 1993; Voiculescu et more of the cell population was transduced by individual re-
al.,2001). combinant retroviruses.The virally transducednonclonal pop-
Krox20, also known as early growth response 2 (Egr2), be- ulationofpuromycinresistantcellswasexpanded,culturedto
longs to the zinc finger-encoding, egr family of genes whose confluence,andharvestedforWesternblotanalysistoconfirm
transcription is rapidly activated by extracellular stimuli in the expressionofthevirallyexpressedgene.
absence of de novo protein synthesis. EGR proteins relay ex- InthecellsinfectedwiththeKrox20-expressingretrovirus,a
tracellular signals and elicit long-term intracellular responses unique 48 kDa band was detected, while, in the PPARγ-line,
by regulating the transcription of their target genes through the 60 kDa PPARγ protein was detected. Neither protein was
specific binding to a GC-rich region (Swirnoff and Milbrandt,
detectable in the control line (Figure 1A). RT-PCR was also
1995).EGRproteinsplayimportantrolesincellulargrowthand
used to confirm the presence of viral transcripts (data not
differentiation and responses to hormonal stimuli (Lee et al.,
shown).Eachofthetransducedcelllineswastheninducedto
1996; Swiatek and Gridley, 1993; Schneider-Maunoury et al.,
differentiateuntilday7.OilRedO(ORO)stainingofthedishes
1993; Topilko et al., 1994; Tourtellotte and Milbrandt, 1998).
atdifferenttimepointsdemonstratedthatKrox20-overexpress-
Forexample,Egr1,alsoknownasKrox24,hasbeenidentified
ingcellsbegantoaccumulatelipidearlierthancontrolcells.In
as the transcription activator of low-density lipoprotein recep-
addition,after7days,ahigherpercentage(around90%)offat-
torinresponsetooncostatinMstimulation(Zhangetal.,2002).
IthasalsobeenestablishedthatKrox20expressionisessential laden cells was evident, as compared to the control cell line
forthefinaldifferentiationofmyelinatingSchwanncells(Zorick (up to 70%). The fat droplets in differentiated cells in the
etal.,1996;Warner etal.,1998).However,arole ofKrox20in Krox20 line also appeared larger. The differentiation of the
thefatcelldevelopmenthasnotbeenpreviouslyexplored. PPARγcellswasevenmorerobust,withnearlyallofthepreadi-
TheaimofthesestudieswastoinvestigatewhetherKrox20 pocytesdevelopingintoadipocyteswithlargefatdroplets(Fig-
playedaroleintheprocessofadipocytedifferentiationandto ure 1B). Consistent with these morphological observations,
further provide insights into its mechanism. Our findings have Krox20 enhanced the expression of aP2, adipsin, and PPARγ,
uncoveredanessential,stimulatoryroleofKrox20inadipogen- markergenesforadipogenesis,withasignificantlyhigherlevel
esisinvitropartiallythroughandincooperationwithC/EBPβ. ofexpressionandwithafasterkineticsthaninthecontrolcell
line(Figure1C).Thesedataindicatethat,asassessedbyboth
Results morphologic and molecular criteria, overexpression of Krox20
stimulatesadipogenesisin3T3-L1preadipocytes.
Krox20isanimmediate-earlygene
during3T3-L1adipogenesis
Krox20cancommitthemultipotentNIH3T3
WehavepreviouslyshownthatKrox20ishighlyenrichedinthe
cellstoanadipogenicfate
white adipose tissue in vivo but not readily detectable in fully
The high level of expression of Krox20 in vivo suggested that
differentiated adipocytes cultured from 3T3-L1 cells in vitro.
However, because Krox20 has been shown to be transiently itmightalsobeabletostimulatedifferentiationinNIH3T3cells,
upregulatedinnaiveNIH3T3cellfibroblastsbyserumstimula- alinewithverylittleadipogenicpotential.Totestthis,NIH3T3
tion, we performed a detailed time course of Krox20 expres- cellswereinfectedwithretrovirusescarryingeitherKrox20-HA,
sion during adipocyte development from 3T3-L1 cells in vitro, vector-HA,orPPARγ;selectedwithpuromycin;andexpanded.
focusingonanalysesatearliertimepointsthanwereanalyzed Western blotting was performed to analyze expression of the
in our previously published survey. 3T3-L1 cells were induced transduced gene in each cell line (Figure 2A). All cells were
to differentiate into adipocytes by treating confluent cells with cultured to confluence and then exposed to a similar cocktail
a standardized hormonal cocktail as previously described. asusedwith3T3-L1cells(5(cid:1)g/mlinsulin,1(cid:1)g/mldexametha-
Quantitative RT-PCR (Taqman assay) of Krox20 as well as a sone, 0.5 mM IBMX). At day 7 postinduction, lipid-containing
group of other adipocyte marker genes revealed that Krox20 cells were not detected in the vector control cells, whereas a
mRNAwasfirstinducedat15minafterexposuretotheinduc- significant fraction of the NIH-PPARγ and NIH-Krox20 cells
tion cocktail and peaked around 1 hr postinduction. Krox20
(around 70% and 20%, respectively) differentiated into fat-
mRNAwasquicklydiminishedafterthisandbecameundetect-
ladencellswiththetypicalmorphologyofculturedadipocytes.
able at 6 hr and thereafter (see Figure S1 in the supplemental
TheadditionofthePPARγligand,troglitazone,furtherenhanced
data available with this article online). The kinetics of Krox20
adiposeconversionintheNIH-PPARγandNIH-Krox20-HAcells,
induction was similar to that of C/EBPβ and was expressed
again with noevident changes in the control cells(Figure 2B).
earlier than C/EBPα and PPARγ, two established inducers of
The RNA levels for adipocyte marker genes (aP2, adipsin,
adipocytedifferentiation,suggestingthatitmightplayanearly
PPARγ) were extremely low at day 7 after exposure to the in-
roleintheprocessofadipogenesis.
ductioncocktailinthecontrolline.Incomparison,theseadipo-
EctopicexpressionofKrox20promotesadipogenesis cyte marker genes were all significantly induced in the NIH-
We next tested whether a gain of Krox20 can promote adipo- Krox20cellstolevelsw30%–40%relativetoNIH-PPARγcells
cyte differentiation by examining the effect of constitutive ex- (Figure2C)ThisresultdemonstratedthatexpressionofKrox20,
pression of Krox20 in 3T3-L1 preadipocytes. Cell lines stably likePPARγ,issufficienttoconvertnaiveNIH3T3fibroblastsinto
expressing a HA-tagged Krox20 or a vector-HA control were differentiated adipocytes, an effect that has been only seen
established by retroviral transduction followed by puromycin previouslywithC/EBPsandPPARγ(Yehetal.,1995;Tontonoz
selection. A PPARγcell line was alsoestablished for compari- etal.,1994).
94 CELLMETABOLISM:FEBRUARY2005
Krox20isanearlykeyfactorinadipogenesis
Figure1.Krox20promotesadipogenesisinthe3T3-L1preadipocyte
3T3-L1cellswereinfectedwithrecombinantretrovirusesderivedfrompMSCVpuro-HA,pMSCVpuro-Krox20-HA,orpMSCVpuro-PPARγ;selected;andinducedfor
differentiationtillday7.
A)TotalproteinlysateswereharvestedatconfluenceforWesternblotanalysisshowingtheoverexpressedKrox-HAorPPARγproteins.Equalamountsoftotallysates
wereblottedwithanti-β-actinasacontrol.
B)OROstainingofindividualcelllinesatdays3,5,or7showingfatdevelopment.
C)TaqmanassaywasperformedtoanalyzethemRNAlevelsofaP2,adipsin,andPPARγatdifferenttimepointspostinductionasindicated.
CELLMETABOLISM:FEBRUARY2005 95
A R T I C L E
Figure2.Krox20inducesadipogenesisinthenaiveNIH3T3cells
NIH3T3cellswereinfectedwithrecombinantretrovirusesderivedfrompMSCVpuro-HA,pMSCVpuro-Krox20-HA,orpMSCVpuro-PPARγ;selected;andinducedfor
differentiationwithahormonalcocktailwithorwithouttroglitazoneuntilday7.
A)WesternblotanalysisofthecellsatconfluenceshowingectopicexpressionofKrox-HAorPPARγ.Equalamountsoftotallysateswereblottedwithanti-β-actinas
acontrol.
B)(Toppanel)AmacroscopicviewoftheORO-staineddishesonday7.(Bottompanel)Amicroscopicview.Scalebars,100(cid:1)M.
C)TaqmanassaywasperformedtoanalyzethemRNAlevelsofaP2,adipsin,andPPARγatday7.
96 CELLMETABOLISM:FEBRUARY2005
Krox20isanearlykeyfactorinadipogenesis
Krox20genesilencingusingsiRNAresults cumulationwasmarkedlyreducedintheKrox20andthecon-
inimpairedadipogenesis trol lines. In contrast, there was still considerably more lipid
TodeterminewhetherKrox20isnecessaryfor adipogenesis,we accumulation in the PPARγ line than in the control line, even
nextusedretrovirusescarrying smallinterferingRNA(siRNA)for without DEX or IMBX. Thus, the promoting effect of Krox20
Krox20toknockdownitsexpression.Twoindependenthairpin wasdependantonDEXand/orIBMX,whichareknowninduc-
siRNA oligonucleotide sequences from Krox20 were selected ersofC/EBPδandC/EBPβ,respectively(Caoetal.,1991).Fi-
andclonedintothepSIREN-RetroQretroviralplasmid.There- nally,theKrox20linedidnotaccumulateappreciablymorelipid
combinant plasmids were transfected into the Phoenix pack- thanthecontrollinewhentroglitazonewasaddedalone(con-
aging cells to produce retroviruses that carry both the siRNA dition E) or in the presence of standard culture media without
hairpinandthepuromycinresistancegene.Anegativecontrol INS,DEX,orIBMX(conditionF).Taqmanassaywasperformed
oligonucleotidewasprocessedinasimilarmanner. to examine the mRNA levels of aP2, adipsin, and PPARγ at
3T3-L1cellswereinfectedwiththehairpin-containingviruses, eitherday0orday7underthesevariousconditions(A–F),and
selected,andestablishedasastablecelllinewithconstitutive theresultswereconsistentwiththemorphologicchanges(Fig-
siRNA expression. Cells were cultured to confluence and ex- ure4C).Takentogether,thesedatasuggestthattheadipogen-
posedtothestandarddifferentiationcocktail.TotalRNAatdif- esis-promotingeffectofKrox20isdependentonthepresence
ferenttimepointsduringtheearlytimewindowwascollected, ofDEXandIBMX.
andthemRNAlevelofKrox20andothergeneswasdetermined
byTaqman assay.At30 minpostinduction,the si-Krox20-aand
EctopicexpressionofKrox20leadstoaninduction
si-Krox20-b lines showed slightly lower levels of Krox20 ex- ofC/EBPβexpressionandtransactivation
pression than the control line; at 1 hr, a time when Krox20 ofC/EBPβpromoter
mRNA expression is maximal, the si-Krox20-a and si-Krox20-b Krox20 and C/EBPβ are expressed with similar kinetics, and
lines displayed less than 30% of the expression level of the
both promote adipogenesis, suggesting that they might both
control line.These data showedthat two differentsiRNA hair-
be elements of a differentiation pathway that regulates early
pinsresultedinanefficientknockdownofKrox20(Figure3A).
cellular programs that promote adipogenesis. To further ad-
Thecelllineswerethenculturedinthepresenceofthediffer-
dressthispossibility,wefirstcomparedtheexpressionlevelof
entiationcocktailfor8days.OROstainingofthedishesatdays
C/EBPβin3T3-L1-Krox20-HAcellswiththatofthe3T3-L1-HA
4and8demonstratedthatthesi-Krox20-aandsi-Krox20-blines
controlcells.TotalRNAofthesetwocelllineswascollectedat
accumulated significantly less lipid than the control line (Figure
different time points in the early window postinduction under
3B).MeasurementoftheadipocytegeneRNAlevelsbyTaqman
conditionsA–Dasdescribedabove(seeFigure4).Whencells
assayconfirmedthatthedifferentiationprocessinthesi-Krox20-a
wereexposedtoafullcomplementofhormones(conditionA),
and si-Krox20-b lines was suboptimal and much less robust
C/EBPβ mRNA was increased w3 fold in Krox20-expressing
than the control line (Figure 3C). At day 8, the RNA levels of
aP2, PPARγ, and adipsin in the si-Krox20-a or -b lines were cellsascomparedtothecontrolline.C/EBPβwasstillinduced
w2foldintheKrox20cellswheninsulinwasomitted(condition
reducedto30%–50%ofthoseincontrolcells.However,thesi-
B).WheneitherDEXorIBMXwasdeprived(conditionsCorD),
Krox20-aandsi-Krox20-bsiRNAsdidnotcompletelyabrogate
therewasonlymarginalincreaseinC/EBPβmRNAinKrox20-
adipogenesis, perhaps due to the residual level of Krox20 ex-
expressingcellsascomparedtothecontrolline(Figure5A).
pression. These results establish that Krox20 is necessary for
We further harvested total protein lysates of each cell line
normaldifferentiationof3T3-L1cells.
under various conditions at day 2 postconfluence and per-
formed Western blotting to examine C/EBPβ protein levels. In
Systematicdeprivationofhormonalcomponents
compromisestheabilityofKrox20 conditions A and B, signal was much stronger for C/EBPβ in
topromoteadipogenesis the Krox20 cells than in the control cells, whereas, in condi-
WenexttestedwhetherKrox20canobviatetheneedforsome tions C and D, increase in C/EBPβ protein expression was
orallofthecomplementofhormonesnecessaryfortheinduc- barely detected (Figure 5B). These data indicate that ectopic
tionofadipogenesis.Forexample,PPARγ(butnootherfactors) expression of Krox20 leads to an induction of C/EBPβ mRNA
can fully induce adipocyte differentiation in the absence of andproteininaDEX-andIBMX-dependentfashion.
these hormonal factors. Krox20, PPARγ, and control cell lines Theaboveresultspromptedustofurtherinvestigatewhether
were established as described and confirmed to express the Krox20 would transactivate C/EBPβ promoter. For this pur-
virallytransducedgenes(Figure4A).Cellsweregrowntocon- pose,the pMSCVpuro-Krox20-HAorthe controlpMSCVpuro-
fluence and treated with an adipogenic cocktail in which dif- HAplasmidswerecotransfectedwithaC/EBPβpromoterlucif-
ferent components were systematically omitted. A separate erase reporter plasmid (B3K) (generously provided by Dan
condition using a PPARγ ligand (troglitazone) alone was in- Lane)into3T3-L1cellsat80%confluence(Zhangetal.,2004).
cluded. The cells were then cultured for 7 days when ORO The empty reporter vector (BA) was included as a control. At
stainingwasperformed(Figure4B).Fulldifferentiationwasob- day2postconfluence,cellsweretreatedwiththedifferentiation
served for all cell lines when insulin (INS), dexamethasone cocktail or media alone. Protein lysates were harvested, and
(DEX),andIBMXwereallincluded(conditionA).WhenINSwas luciferase assays were performed 12 hr later (Figure 5C). Co-
omitted (condition B), there was a modestly decreased fat transfection of Krox20 transactivated the C/EBPβ promoter
content in each cell line, but Krox20 still exerted a significant (B3K)3-foldtothatseenwiththecontrolpMSCVpuro-HAplas-
inductive effect. This indicates that INS is dispensable for mid; omission of the hormonal cocktail abrogated this effect,
Krox20’s promoting effect. In contrast, when either DEX or suggestingthattransactivationofC/EBPβbyKrox20isdepen-
IMBX was removed (conditions C and D, respectively), fat ac- dentonhormonalinducers.
CELLMETABOLISM:FEBRUARY2005 97
A R T I C L E
Figure3.EffectofsiRNAknockdownofKrox20on3T3-L1adipogenesis
3T3-L1cellswereinfectedwithpSIREN-RetroQ-derivedretrovirusescarryingeithersiRNAoligosforKrox20(si-Krox20-aor-b)orcontrololigo,selectedforpuromycin
resistance,andexpanded.Cellsweretheninducedbyregularhormonalcocktailtodifferentiatetillday8.
A)TaqmanassaywasperformedtoanalyzetheexpressionlevelofKrox20atdifferenttimepoints,asindicatedintheearliestwindowofinduction.
B)OROstainingofindividualdishesatday4orday8showingfatdevelopment.
C)TaqmanassayshowingexpressionlevelsofaP2,adipsin,andPPARγatdifferenttimepointsduringdifferentiation.
98 CELLMETABOLISM:FEBRUARY2005
Krox20isanearlykeyfactorinadipogenesis
Figure4.Theadipogenesis-promotingeffectofKrox20isreducedbysystematicdeprivationofnormalhormonalstimulants
Stable3T3-L1celllinesthatweretransducedwithKrox20-HA,PPARγ,orcontrolwereestablishedasinFigure2andinducedfordifferentiationwithhormonalcocktails
asindicatedtillday7.
A)WesternblotanalysesofthecellsatconfluenceshowingectopicexpressionofKrox20orPPARγproteins.Equalamountsoftotallysateswereblottedwithanti-β-
actinasacontrol.
B)OROstainingofindividualdishesatday7showingfatdevelopment.
C)TaqmanassaywasperformedtoanalyzethemRNAlevelsofaP2,adipsin,andPPARγunderdifferentconditionsatdays0(confluence)or7.
CELLMETABOLISM:FEBRUARY2005 99
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Figure 5. Ectopic expression of Krox20 enhances
C/EBPβexpressionandtransactivatestheC/EBPβ
promoter
A)The3T3-L1-Krox20-HAorcontrolcelllinesfrom
Figure4wereinducedtodifferentiateundercondi-
tionsA–DasdescribedinFigure4.TotalRNAwas
harvestedatdifferenttimepointspostinductionas
indicated. Taqman assay wascarried out to mea-
suretheC/EBPβmRNAlevel.
B)Proteinlysatesforbothcelllinesunderallfour
conditions werecollected at 2days postinduction
foranti-C/EBPβWesternblot(upperpanel);anti-β-
actinblotwasincludedascontrol(lowerpanel).
C)pMSCVpuro-Krox20-HAorpMSCVpuro-HAwas
cotransfectedinto3T3-L1preadipocyteswithindi-
vidualreportersasdenoted,alongwiththepRL-TK
as an internal control. Cells were induced to dif-
ferentiate2dayslater.Totalproteinswereharvested
at12hrpostinductionanddualluciferaseactivities
determined.Thevalueoffireflyluciferasewasnor-
malizedbytherennilaluciferasetogeneratetherel-
ative luciferase activity; error bars represent the
standard deviation of three independent experi-
ments.
Krox20exertsitsadipogeniceffectatleastinpart oligos targeting C/EBPβ (si-C/EBPβ-a and si-C/EBPβ-b), to-
throughactivatingC/EBPβ gether with a hygromycin resistance-carrying retrovirus that
The above results suggested that Krox20 might promote adi- encodes Krox20-HA. Control lines were also established. All
pogenesisbyactivatingC/EBPβ.Wethereforeexaminedwhether lines were induced for 7 days. At day 2, protein lysates were
reduced expression of C/EBPβ by siRNA would compromise harvested,andthelevelofvirallyexpressedKrox20(Figure6A,
theinductiveeffectofKrox20onadipogenesisin3T3-L1cells. middle panel) and endogenous C/EBPβ proteins were deter-
3T3-L1cellswerecoinfectedwith eitheroftwodifferentpuro- minedbyWesternblotting(Figure6A,upperpanel).
mycin resistance-carrying retroviruses that deliver hairpin Inthecontrollines(Figure6A,lanes1–3)andKrox20-overex-
100 CELLMETABOLISM:FEBRUARY2005
Krox20isanearlykeyfactorinadipogenesis
Figure6.siRNAknockdownofC/EBPβpartiallyimpairsthepromotingeffectofKrox20
3T3-L1cellswerecoinfectedwithpMSCVhyg-derivedretrovirusescarryingeitherKrox20-HAorcontrolinsertandpSIREN-RetroQ-derivedretrovirusescarryingeither
siRNAoligosforC/EBPβ(si-C/EBPβ-aand-b)orcontrololigo,selectedforhygromycinandpuromycinresistance,andinducedtodifferentiatetillday7.
A)Westernblotanalysisofindividualcelllinesatday2showingendogenouslevelofC/EBPβ(toppanel)andtheectopicallyexpressedKrox20(middlepanel).Anti-β-
actinblotwasincludedascontrol(bottompanel).
B)OROstainingofindividualdishesatday7showingfatdevelopment.
C)TaqmanassayoftheexpressionlevelofaP2,adipsin,andPPARγofdifferentcelllinesattimepointsindicated.
pressing lines (Figure 6A, lanes 4–6), delivery of specific hair- 7 showed that delivery of either si-C/EBPβ-a or si-C/EBPβ-b
pins resulted in a significant reduction of C/EBPβ by at least resultedinasignificantreductionoffatcontent(Figure6B,up-
70%comparedtothenegativecontrololigo,demonstratingan perdishes).Thisisconsistentwithearlierreportsthatalossof
efficient knockdown of C/EBPβ by both the si-C/EBPβ-a and C/EBPβ function reduces adipogenesis in vivo and in vitro
si-C/EBPβ-bhairpins.OROstainingofcontrolcelllinesonday (Hammetal.,2001;Tanakaetal.,1997).Deliveryofeitherhair-
CELLMETABOLISM:FEBRUARY2005 101
A R T I C L E
pin into the Krox20-overexpressing cells also significantly re- and, in cultured cells, C/EBPβ plays a key role in elevating it.
duced fat accumulation (Figure 6B, bottom dishes), showing However, what drives C/EBPβ has been an important unan-
that knockdown of C/EBPβ reduced the proadipogenic effect swered question. Our studies identify Krox20 as an important
ofKrox20.However,Krox20overexpressioninthesi-C/EBPβ-a early regulator that controls C/EBPβ level and complements
orsi-C/EBPβ-blinesstillexhibitedmorefataccumulationthan C/EBPβ in inducing PPARγ and the full program of adipo-
control cells (Figure 6B, comparing upper dishes and bottom genesis.
dishesinlanes1and2).TheeffectoftheC/EBPβknockdown Krox20overexpressionleadstoupregulationofPPARγgene
was evaluated by Taqman assay of aP2, adipsin, and PPARγ expression and augments morphologic differentiation of 3T3-
RNAlevelsatconfluence,day2,andday7(Figure6C).These L1.siRNA-mediatedreductionofKrox20levelreducestheRNA
studies showed that the si-C/EBPβ-a or si-C/EBPβ-b hairpins levels of PPARγ and impairs 3T3-L1 adipogenesis, indicating
reduce but do not fully block the ability of Krox20 to induce that Krox20 is required in this process. During adipogenesis,
adipocytemarkergenes.ThesedatasuggestthatKrox20acts PPARγ functions as a “switch” that regulates whether or not
inpartbyactivatingC/EBPβ,butitmightalsoexertitsproadi- preadipocytesdifferentiateintoadipocytes(Rosenetal.,1999,
pogeniceffectsindependentofC/EBPβ. 2002).TheseresultssuggestthatKrox20isanimportantcom-
ponent of the transcription factor network controlling whether
Krox20cooperateswithC/EBPβininducing ornotPPARγisexpressedinsufficientquantitiestofullyinduce
adipogenesisinNIH3T3cells adipocytedifferentiation.
If Krox20 has inductive effects on adipogenesis that are inde- TheseeffectsofKrox20aremediatedinpartbyinductionof
pendent of C/EBPβ, we predicted that cotransfection of both C/EBPβgeneexpression.C/EBPβhasbeenshownpreviously
genes would have an additive effect even in undifferentiated tobeamongthefirsttranscriptionfactorsactivatedduringadi-
3T3 cells. To test this, naive NIH3T3 cells were infected with pogenesis. Krox20 gene expressionis also induced early dur-
retrovirusescarryingKrox20-HA,C/EBPβ,orbothgenes.Con- ingadipogenesisinresponsetoINS,DEX,andIBMX,withsim-
trol infections with empty vectors were included. Cells were ilar kinetics to C/EBPβ. Increased Krox20 expression results
selected, expanded, and induced to differentiate for 7 days. in upregulation of C/EBPβ gene expression, and Krox20 can
ProteinassaysusingWesternblotsconfirmedtheoverexpres- transactivate the C/EBPβ promoter, suggesting that Krox20’s
sion of C/EBPβ and Krox20 in the virally infected cells (Figure positiveeffectonadipocytedifferentiationmaybemediatedat
7A).OROstainingonday7indicatedthatKrox20andC/EBPβ least in part by transactivation of C/EBPβ. This conclusion is
overexpressioncouldinduceadipogenesisinsomebutnotall further supported by the reduction in differentiation of cells
ofthecells. overexpressingKrox20whenC/EBPβlevelsareknockeddown
Incontrast,whenKrox20andC/EBPβwerecoexpressed,all byRNAi.However,thesestudiesdonotestablishwhetherthe
cells became fully differentiated adipocytes, with a substan- effectsofKrox20onC/EBPβgeneexpressionaredirectorin-
tially greater amount of lipid accumulation in each cell (Figure direct.Inpreliminarystudies,Krox20wasabletotransactivate
7B).Thisinductiveeffectwasindistinguishablefromthatseen aC/EBPβpromoterluciferasereporterconstruct(Figure5),and
withPPARγ(asshowninFigure3).AssaysofthemRNAlevels adeletionseriesfurtherlocalizedtheresponseelementtobe-
confirmed that cells infected with both the Krox20 and C/EBPβ tween−1438and−1134basepairs(datanotshown).Theabil-
viruseshadhigherlevelsofaP2, adipsin,andPPARγthaneither ity of Krox20 to activate the C/EBPβ promoter depended on
gene alone (Figure 7C), further demonstrating that these two thepresenceofthestandardhormonaldifferentiationcocktail,
geneshaveasynergisticeffectonadipogenesisinNIH3T3cells. as was also the case with Krox20’s inductive effect on adipo-
genesis.However,the3kbproximalpromoterofC/EBPβdoes
Discussion not contain a consensus Krox20 response element (T-G-C-G-
T/g-G/A-G-G-C/a/t-G-G/T) (Swirnoff and Milbrandt, 1995). In
Cell specification in mammals is a multistep process in which addition, C/EBPβ promoter sequences were not detected in
cells first become determined or committed to a develop- chromatin immunoprecipitation assays after precipitation of
mentalfate,afterwhichtheydifferentiateandacquiretheprop- Krox20 (data not shown), suggesting that Krox20 activates
erties of a specific cell type. Adipocyte development is con- C/EBPβgeneexpressionindirectly.Inthisregard,thepurifica-
trolled by a series of steps that lead fibroblasts to become tion of the factor(s) binding to C/EBPβ promoter sequences
preadipocytes.Whenfurtherinduced,preadipocytesdifferenti- between −1438 and −1134 may lead to the elucidation of the
ateandexpressgenesthatallowthemtostorelipidandcarry cellularpathwaysresponsiblefortheactivationofC/EBPβdur-
numerousotherregulatoryfunctionsperformedbymatureadi- ingadipogenesis.
pocytes.Whilemanyofthecomponentsofthegeneregulatory The residual adipocyte phenotype in the cells expressing
network that controls the differentiation of adipocytes have Krox20 and the C/EBPβ siRNA suggests that Krox20 also ex-
been elucidated in studies of cultured 3T3-L1 and 3T3-F442 erts its effect via C/EBPβ-independent pathways. This possi-
preadipocytes, recent evidence has suggested that additional bility is further supported by the observation that Krox20 and
factorsarelikelytobenecessaryinvivo(Soukasetal.,2001). C/EBPβ have synergistic effect on the differentiation of naive
To identify such factors, we have studied the functions of a 3T3 cells. Indeed, the expression of both factors together re-
number of transcription factors that are enriched in adipose sults in a fully induced adipocyte phenotype in NIH3T3 cells
tissue in vivo. The results presented here identify Krox20 as similar to that seen after expression of PPARγ. This effect is
playing akey role inthe process of adipogenesisand charac- notevidentwithanyothertranscriptionfactors.Takentogether,
terizeitasoneofthesmallgroupoffactorscapableofinduc- theseobservationssuggestthatKrox20playsanearlyrole,in-
inguncommittedfibroblaststobecomeadipocytes.Ithasbeen deedtheearliestknownrole,infatcelldevelopment.
well established that PPARγ is the really dominant regulator, TheC/EBPβ-independentmechanismbywhichKrox20stim-
102 CELLMETABOLISM:FEBRUARY2005