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http://dx.doi.org/10.10014-4827/& 2014 El

nCorresponding auE-mail addresses1 Tissue Protectio

Research Article

Erg cooperates with TGF-β to controlmesenchymal differentiation

Megan K. Cox1, Brittany L. Appelboom, Ga I Ban, Rosa Serran

Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham,1918 University Blvd, MCLM 660, Birmingham, AL 35294-0005, United States

a r t i c l e i n f o r m a t i o n

Article Chronology:

Received 6 May 2014Received in revised form8 August 2014Accepted 9 August 2014Available online 17 August 2014

Keywords:

Intervertebral discAnnulus fibrosusSclerotomeSmad3Adamtsl2Scleraxis

016/j.yexcr.2014.08.015sevier Inc. All rights reserv

thor.: Megan.Cox@genzyme.comn and Repair, Genzyme Co

a b s t r a c t

Transforming growth factor β (TGF-β) signaling plays an integral role in skeletal develop-ment. Conditional deletion of the TGF-β type II receptor (Tgfbr2) from type II Collagen (Col2a)expressing cells results in defects in development of the annulus fibrosus (AF) of theintervertebral disc (IVD). We previously used microarray analysis to search for marker genes ofAF as well as transcription factors regulated by TGF-β during AF development. The transcriptionfactor avian erythroblastosis virus E-26 (v-ets) oncogene related (Erg) was identified in themicroarray screen as a candidate regulator of AF development. To study the effects of TGF-β on AFdifferentiation and the role of Erg in this process, we used mouse sclerotome grown in micromass

cultures. At 0.5 ng TGF-β/ml, sclerotome cells started to express markers of AF. Regulation of Ergby TGF-β was confirmed in these cells. In addition, TGF-β soaked Affi-gel beads implanted into theaxial skeleton of stage HH 25 chick embryos showed that TGF-β could induce expression of ErgmRNA in vivo. Next, an adenovirus to over-express Erg in primary sclerotome micromass cultureswas generated. Over-expression of Erg led to a change in cell morphology and inhibition ofdifferentiation into hyaline cartilage as seen by reduced Alcian blue staining and decreased Sox9and c-Maf expression. Erg was not sufficient to induce expression of AF markers and expression ofSca1, a marker of pluripotent progenitor cells, was up-regulated in Erg expressing cells. Whencells that ectopically expressed Erg were treated with TGF-β, enhanced expression of specificdifferentiation markers was observed suggesting Erg can cooperate with TGF-β to regulatedifferentiation of the sclerotome. Furthermore, we showed using co-immunopreciptiation that

Erg and Smad3 bind to each other suggesting a mechanism for their functional interaction.& 2014 Elsevier Inc. All rights reserved.

Introduction

Intervertebral disc (IVD) is derived from embryonic structurescalled the sclerotome and notochord [1–3]. The nucleus pulposus,the cushioning core of the mature IVD, is derived from the

ed.

(M.K. Cox), rserra@uab.erporation, 49 New York Av

notochord while the annulus fibrosus (AF), which providesthe structural properties of the IVD, is derived from sclerotome.The sclerotome is derived from somites, transient structures thatdetermine the segmented nature of the embryo [4]. In response tosignals from the notochord and floor plate of the neural tube, the

du (R. Serra).enue, Framingham, MA 01701, United States.

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maturing somites undergo dorsal-ventral compartmentalizationestablishing the dermomyotome and sclerotome, the latter form-ing most of the connective tissues of the future axial skeleton. Thesclerotome is a pluripotent mesenchyme and gives rise to thecartilage of the vertebral bodies, AF of the disc, and tendon. Eachsclerotome segment demonstrates rostral to caudal polarity anddue to resegmentation of sclerotome, the caudal domain of thesclerotome will form the anterior structures of each vertebra andthe rostral domain forms the posterior structures [5,6]. The AFwill form from the cells in the sclerotome adjacent to the borderbetween the sclerotome halves. While many aspects of develop-ment in the axial skeleton have been elucidated, the developmentof the IVD is still poorly understood. Transforming growth factor-beta 3 (Tgfb3) is one of the earliest markers of the developing IVDwithin the sclerotome [7].

TGF-β is a critical component for many aspects of development[8]. TGF-βs signal through heteromeric type I and type II serine/threonine kinase receptors. The current model is that TGF-β bindsto the type II receptor (Tgfbr2) on the cell surface, recruits thetype I receptor (Tgfbr1) to form a heterotetrameric complex, thenTgfbr2 phosphorylates the type I receptor activating the type Iserine/threonine kinase [9]. Smad proteins are phosphorylatedby the type I receptor, translocate to the nucleus and act astranscription factors [10]. In particular, TGF-β signaling is nece-ssary for proper development of the AF of the IVD. Conditionaldeletion of Tgfbr2, results in the disruption of development in theaxial skeleton, including loss of the AF without affecting thenucleus pulposus of the IVD [11,12]. Previously, we used micro-array analysis to identify genes that could be used as markers ofthe AF during development, as well as genes downstream ofTGF-β signaling that would potentially be involved in controllingdifferentiation of the AF [13]. We identified avian erythroblastosisvirus E-26 (v-ets) oncogene related (Erg) as being localized tothe IVD during development, down-regulated by Tgfbr2 deletionin vivo, and up-regulated by TGF-β treatment in sclerotomecultures suggesting that it may play an important role in differen-tiation of the AF from sclerotome.

Erg is a member of the ERG sub group of the ETS family oftranscription factors [14]. These transcriptions factors are allgrouped based on their ETS binding site and have been shownto frequently act as co-transcription factors [15,16]. It has alsobeen shown that ERG can activate a TGF-β response element [17]and that the common fusion proteins seen in prostate cancercan act as dominant negative forms and inhibit expression ofTgfbr2 [17]. During development Erg has been shown to beexpressed in the developing IVD as well as the interzone indeveloping synovial joints [18–23]. A chondrocyte specific form ofERG, known as ERG C-1-1, was identified in chick and when overexpressed blocked hypertrophic differentiation in chondrocytes[20–22]. The role of Erg in development of the AF was notaddressed. We therefore evaluated Erg for its potential to regulateAF differentiation.

In this study, we confirm that TGF-β regulates expression of Ergin sclerotome cultures and in vivo. We used adenovirus vectors toover-express Erg in sclerotome and show that Erg blocks chon-drogenesis in the cultures but is not sufficient to induce expres-sion of markers for AF differentiation. Expression of Sca1, amarker of pluripotent progenitor cells, is up-regulated in Ergexpressing cells suggesting that Erg maintains cells in a pluripo-tent state. We then show that Erg can cooperate with TGF-β

to stimulate expression of Adamtls2 and Scx, markers of AF andtendon respectively. Furthermore, Erg protein associates withSmad3 in sclerotome cells. We propose that Erg cooperates withTGF-β in the development of the IVD, promoting AF geneexpression and preventing the formation of vertebral cartilagein the IVD space.

Methods

Sclerotome cell isolation and culture

Cells were isolated from wild type (Bl/6 X ICR) e11.5 mouseembryos as described in [13]. Cells were plated in 20 ul drops at adensity of 1.5�107 cells/ml. Drops were allowed to set for 1 hbefore flooding the well with media. Cells were treated withTGFβ1 or BMP the following morning. For experiments withadenovirus, cells were resuspended in media containing 50 MOIadenoviruses before being placed in micromass culture. TGF-β1or BMP4 treatments were performed 48 h after transduction.Cells were fixed with 4% PFA then stained with 1% Alcian blue in0.1 N HCl 3 or 7 days post-transduction. All animals used wereapproved by the University of Alabama at Birmingham's Institu-tional Animal Care and Use Committee.

Adenovirus production and transduction

Full-length mouse Erg was cloned into pEC3.1 which contains aCMV promoter and an IRES- Gfp flanked by attL sites. This vectorwas then recombined using the Gateway Cloning system intopAdBlockIt (Invitrogen). Virus was produced in HEK 293A cells(Invitrogen) following the manufacturer's protocol. Virus wasconcentrated by pelleting the cells and resuspending in 1 ml ofmedia before freeze thawing according to the manufacturer'sprotocol. Virus was titered by infecting 293A cells with serialdilutions of viral supernatant. After 48 h, the number of GFP-positive cells was counted and concentration calculated based ondilution. Control virus contained an additional Gfp in place of Erg.

Chick bead implants

Specific pathogen free, white leghorn fertilized chicken eggs wereobtained from Charles River Labs and incubated at 39 1C. Embryoswere staged according to Hamburger [24]. AffiGel Blue beads100–200 mesh (Bio-Rad) were soaked in 10 ng/ul porcine TGF-Β(RD Systems) or 10 ng/ul BSA for 1 h. The beads were thenimplanted in forelimb and lower spine of HH 23–25 chick embryos.Chick embryos were harvested after 24 h for in situ hybridization.

In situ hybridization

Whole mount in situ hybridization was performed on stage HH 27chick embryos 24 h after implantation. Embryos were fixed in 4%PFA. Embryos were then stored in 100% methanol at �20 1C. Theprobe for the chicken Erg was a kind gift from Iwamoto [20] andwas labeled with digoxigenin. In situ hybridization was performedas described in Riddle et al. [25]. BM Purple (Roche) was used tovisualize alkaline phosphatase label.

Rev

erse

prim

er(5

0 –30)

Pro

duct

size

(bp)

CCGTC

GGTA

CTTG

ACC

ACT

199

TCCC

CGTT

GGTG

CCTT

CCCA

217

GCT

GCT

GATG

GAGAACT

CATT

72GGGGCT

GTA

CTGCT

TAACC

AG

142

TCATC

CAGTA

GTA

GTC

TTCC

AGG

173

TAGGAGGGCA

GATG

GGTA

AGCA

AA

85ACT

CTTC

AGTG

GCA

TCCA

CCTT

CA13

0GATT

GCC

CAGAGTG

CTCG

CCC

129

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qRT-PCR

RNA samples were collected from sclerotome micromass cultures,using RNeasy Plus (QIAGEN). Real time PCR was done usingQuantiFast SYBR Green RT-PCR Kit (QIAGEN) run on a Lightcycler480 (Roche). Primers were designed using NCBI primer BLAST orselected from Primer Bank [26,27] (Table 1). Differential expres-sion and statistical significance were analyzed using REST 2009software (QIAGEN) which allows for efficiency correction as wellas rigorous statistical testing of non-parametric data [28]. RESTuses a pair-wise fixed reallocation randomization test to deter-mine significance. HPRT was used as the normalization control inall experiments. Expression was determined relative to theuntreated or Ad-GFP infected, untreated controls as indicated.

Western blot, immunoprecipitation-western blot analysis

For western blots, 10 ug of protein was run on a 10% SDS-PAGE gelthen transferred to a PVDF membrane. ERG was detected withanti-ERG (Abcam #77258) 1:1000 and GAPDH (Santa Cruz #sc-20357) was used as a loading control.For immunoprecipitation (IP)- western blot assays, protein

samples were collected using lysis buffer (20 mM Tris pH8.0,150 mM NaCl, 2 mM EDTA pH8.0, 10% Glycerol) with 1% TritonX-100 and processed according to standard protocols. Since Erg,Smad3 and IgG heavy chain are all close to 50 kDa, we usedantibodies that were made in different species for the immuno-precipitation and the immunoblot to minimize the detection ofthe IgG heavy chain. Goat anti-ERG (Abcam #77258), Rabbit anti-ERG (Abcam # 92513), and Rabbit anti-Smad3 (Cell Signaling#9513) antibodies were used as indicated. Secondary antibodiesused were HRP conjugated anti Rabbit IgG (Santa Cruz #B2213)and HRP conjugated anti Goat IgG (Santa Cruz #G0208).

Table1–List

ofpr

imersusedforQRT-PCR

.

Gen

eNam

eFo

rwardpr

imer

(50 –30)

Ada

mtsl2

GGGCA

ACA

ATC

ATC

TTGGTT

ACT

Erg

TCACC

CCTC

AGTC

CAAAGCT

GC

Fmod

CCTC

CTGTC

AACA

CCAACC

THprt1

TCAGTC

AACG

GGGGACA

TAAA

Maf

GGAGACC

GACC

GCA

TCATC

Sca1

TGTG

TTACT

CAGGAGGCA

GCA

GTT

Scx

TCTG

CCTC

AGCA

ACC

AGAGAAAGT

Sox9

GGCC

GAAGAGGCC

ACG

GAAC

Results

TGF-β induces expression of Erg, an AF enrichedtranscription factor

Primary sclerotome cells grown in micromass in the absence ofgrowth factors demonstrate formation of Alcian blue stainednodules containing round cells representing hyaline cartilage,similar to what is seen in cultures of limb mesenchyme (Fig. 1A)[29]. We previously showed that sclerotome cultures treated with5 ng TGF-β/ml demonstrated a reduction in Alcian blue stainednodules. In addition, the cells in the culture were not round andtook a spindle shape [13]. Others have recently observed a similarresponse to TGF-β in limb mesenchyme grown in micromassculture [29–31]. To further characterize the effects of TGF-β onsclerotome micromass cultures we treated cells with varyingconcentrations of TGF-β1 or BMP4 (Fig. 1A). BMP is a knownchondrogenic factor [32] and treatment with BMP resulted in adose dependent increase in Alcian blue stained nodules withhyaline cartilage morphology (Fig. 1A). Treatment with varyingdoses of TGF-β resulted in a dose dependent decrease in Alcianblue stained nodules (Fig. 1A). We previously showed an increasein markers for AF in sclerotome treated with 5 ng TGF-β/ml [13].Here we confirm up-regulation of Fibromodulin (Fmod) andAdamtsl2 by varying doses of TGF-β in sclerotome cultures

Fig. 1 – TGF-β and BMP4 act in a dose-dependent fashion to regulate differentiation in sclerotome cells. (A) Alcian blue staining ofmicromass cultures treated with varying doses of TGF-β1 or BMP4 for 5 days. (B, C) Quantitative real-time RT PCR of AF enrichedgenes up-regulated by TGF-β1 (B) and genes down regulated or unchanged (C) by TGF-β1 treatment. Data analyzed by REST.Expression is relative to untreated cells, which are set at 1.0 (dashed line). HPRT was used as the normalization control. Asterisks(���) with a horizontal line indicates po0.05 relative to untreated controls. The step lines indicate po0.05 between the twosamples connected, n¼4.

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(n¼4 separate experiments) using quantitative real time RT-PCR(Fig. 1B) where expression was determined relative to untreatedcontrols and HPRT was used as the normalization control.Adamtsl2 was regulated in a dose-dependent manner between0.5 and 5 ng TGF-β/ml; however, maximal expression of Fmodwas observed at the lowest dose tested. Scleraxis (Scx), a markerof tendon differentiation was also up-regulated in a dose depen-dent manner. It was previously shown that TGF-β regulatestendon differentiation in culture and in vivo [33]. Sox9, one ofthe master regulators of cartilage differentiation, was not regu-lated by TGF-β at any dose tested whereas, c-Maf, a transcriptionalpartner of Sox 9 [34] was down regulated by TGF-β (Fig. 1C). Sca1,a marker of pluripotent mesenchymal stem cells [35], was notregulated by TGF-β (Fig. 1C). The results confirm previous studiesindicating TGF-β promotes a fibrous phenotype in limb mesench-ymal cells and extend the studies to sclerotome [13,29,31].

Using microarray screens, we previously identified Erg as atranscription factor that was enriched in the AF and regulated insclerotome by TGF-β [13]. To verify regulation of Erg by TGF-βwe measured Erg mRNA levels in sclerotome cultures treated with5 ng TGF-β/ml for 24 h (Fig. 2A; n¼7 separate experiments). TGF-β

treatment resulted in a significant increase in Erg mRNA levels in thesclerotome cultures. To determine if TGF-β could regulate Erg in anin vivo model system, we implanted TGF-β soaked Affy-blue beadsin chicken embryos in ovo and looked for induction of Erg mRNA bywhole mount in situ hybridization. Beads soaked in bovine serumalbumin (BSA) were used as controls. After 24 h with TGF-β, we sawincreased Erg mRNA expression in both the limb bud (Fig. 2B and C,arrows) and spine (Fig. 2D–F, arrows) compared to the BSA controls.The results indicate that TGF-β regulates expression of Erg in bothcell culture and in vivo models.

ERG is not sufficient to induce expression of AF markergenes

Since Erg was localized to the AF and regulated by TGF-β, wetested the hypothesis that Erg, like TGF-β, would regulate expres-sion of markers for AF in sclerotome cells. To test this hypothesis,we generated an adenovirus vector that would express mouseErg (Ad-Erg) when transduced into sclerotome cells in culture.An adenovirus expressing only GFP (Ad-GFP) was used as acontrol. Cells were infected with Ad-GFP or Ad-Erg for 48 h at

Fig. 2 – TGF-β stimulates Erg expression. (A) Quantitative real-time RT PCR showing Erg expression in sclerotome micromasscultures after treatment with 5 ng TGF-β1/ml for 24 h. Data analyzed by REST. Expression is relative to untreated cells set at 1.0.HPRT was used as the normalization control. Asterisks (�) indicates po0.05, n¼7. (B–F) In situ hybridization for Erg 24 h post-implantation of TGF-β1 (C, E, F) or BSA (B, D) soaked bead in the forelimb (B, C) or lower spine (D–F) of HH25 stage chick embryos.Arrows denote ectopic Erg expression. Asterisks indicate location of the bead.

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which time RNA and protein were isolated from the cells toconfirm increased expression of Erg by RT-PCR and western blot(Fig. 3A and B). Both mRNA expression (Fig. 3A) and protein levels(Fig. 3B) of Erg were dramatically increased by transduction withad-Erg. Overexpression of Erg in mouse sclerotome micromasscultures for one week resulted in inhibition of Alcian blue staining(Fig. 3C and D). Very few chondrocytic nodules were observed.The Erg cells appeared spindle shaped. The loss of cartilagenodules and spindle shaped cells could also be seen using phasecontrast microscopy (Fig. 3E and F) and with Toluidine blue Ostaining of sections through the cultures (Fig. 3G and H).The loss of Alcian blue stained cartilage nodules and spindle

shaped cells lead us to hypothesize that Erg could promote AFdifferentiation in the sclerotome cultures. RNA isolated from Ad-GFP and Ad-Erg infected cells was used in real time RT-PCR tomeasure the expression of hyaline cartilage and AF enrichedgenes (Fig. 4). Gene expression relative to the Ad-GFP infected,untreated controls was determined. HPRT was used as a normal-ization control. Consistent with the reduction in Alcian bluestained cartilage nodules observed, expression of Erg resulted indown-regulation of Sox9 and c-Maf, markers of chondrocytedifferentiation [36]. Nevertheless, Erg was not sufficient tostimulate expression of Fmod or Adamtsl2, markers of AF, orScx, a marker for tendon differentiation (Fig. 4). Likewise, we didnot detect changes in expression of smooth muscle actin, whichwould indicate smooth muscle differentiation, or Pecam, a markerfor endothelial cell differentiation (data not shown). However,increased expression of Sca1, a marker of pluripotent mesenchy-mal stem cells [35], was observed (Fig. 4). The results suggestedthat in the absence of other factors Erg may act to maintain themesenchymal cells in a pluripotent state.

Erg and TGF-β cooperate to regulate expression of specificgenes.

Since Erg appeared to maintain cells in a pluripotent state, wetested the hypothesis that this would make the cells morecompetent to respond to TGF-β. To this end we infected cellswith either the Ad-GFP or Ad-Erg virus and then treated the cellswith varying concentrations of TGF-β. Cells infected with thecontrol Ad-GFP virus and treated with TGF-β demonstrated a dosedependent decrease in Alcian blue stained nodules as previouslyshown (data not shown). Expression of specific marker genesrelative to untreated, Ad-GFP infected cells (reference set to 1.0)was compared in Ad-GFP infected cells treated with 1 ng/TGF-β/ml,untreated Ad-Erg infected cells, and Ad-Erg infected cells treatedwith 1 ng/TGF-β/ml for 24 h (Fig. 5A and B; n¼4 separate experi-ments). HPRT was used as a normalization control. Markers of AF,Fmod and Adamtsl2, were up-regulated by TGF-β and unaffectedby Ad-Erg alone, as expected. However, expression of Ad-Ergand TGF-β treatment together resulted in a significant increase inAdamtsl2 expression above that observed with TGF-β treatmentalone suggesting cooperation between Erg and TGF-β signaling inthe regulation of Adamtls2 (Fig. 5A). In contrast, Erg did not appearto contribute to regulation of Fmod mRNA (Fig. 5A). Similar toAdamtls2, Erg and TGF-β also appeared to cooperate to regulate theexpression of the tendon marker, Scx (Fig. 5A). Likewise, down-regulation of c-Maf mRNA was significantly greater in Ad-Erginfected cells treated with TGF-β than in untreated Ad-Erg infectedcells suggesting that Erg and TGF-β cooperate to down-regulatec-Maf mRNA (Fig. 5B). Sox9 mRNA levels were not regulated byTGF-β and down-regulated by Ad-Erg as we showed previously.Sox9 was still significantly down regulated in TGF-β treated Ad-Erg

Fig. 3 – ERG inhibits chondrogenesis in sclerotome cells. (A) Quantitative real-time RT PCR was used to verify over-expression ofErg mRNA 3 days post-transduction. Two days of TGF-β treatment did not further regulate Erg mRNA levels in Ad-Erg infected cells.Expression relative to untreated Ad-GFP transduced cells is shown (set to 1.0, dashed line). RT-PCR data was analyzed using REST.HPRT was used as the normalization control. Asterisks (�) indicates po0.05, n¼7. (B) Western blot was used to confirm increasedErg protein levels in Ad-Erg compared to Ad-GFP infected cells at 3 and 4 days post-transduction. GapDH was used as a loadingcontrol. (B–H) Sclerotome micromass cultures were infected with Ad-GFP (C, E, G) or Ad-Erg (D, F, H) for seven days. Alcian bluestaining (C, D), phase contrast images (E, F), and Toluidine blue O staining (G, H) all show a reduction in cartilage nodule formation.

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expressing cells although to a lesser extent than with Ad-Erg alone(Fig. 5B). Sca1, which is regulated by Ad-Erg but not by TGF-β wasstill up-regulated in TGF-β treated Ad-Erg infected cells although toa lesser degree, possibly due to differentiation induced with TGF-βtreatment (Fig. 5B). Together the results suggest that TGF-β andErg cooperate to regulate specific markers for AF and tendondifferentiation.

Since Smad3 is the primary signaling protein for TGF-β, actingas a transcription factor when activated after ligand–receptorbinding, we next tested the hypothesis that Erg and Smad3could participate in a protein complex that would potentiallymediate the functional cooperation observed. To test this hypoth-esis, immunoprecipitation followed by western blot was used(Fig. 6). First, primary sclerotome cells were infected with Ad-Ergor Ad-GFP virus. Smad3 was immunoprecipitated from proteinlysates generated from the infected cells. Immuno-complexeswere resolved on an SDS-polyacrylamide gel and the proteinswere transferred to nitrocellulose membranes, which were incu-bated with antibody directed to the Erg protein followed by asecondary antibody for detection. A large amount of the ectopicErg protein was observed in the immunocomplexes that werepulled down with the Smad3 antibody indicating that Smad3 can

form a complex with Erg (Fig. 6A). Expression of Ad-Erg wasconfirmed by western blot using an aliquot of protein samplesbefore immunoprecipitation (Fig. 6A, input). Complexes of endo-genous Erg and Smad3 could also be pulled down with the anti-Erg antibody (Fig. 6B). The results indicate that Smad3 and Ergcan participate in the formation of protein complexes.

Discussion

Having previously shown that TGF-β signaling pathways play arole in IVD development [12], we focused on transcription factorsregulated by TGF-β as potential mediators of AF differentiation.From an extensive list of genes expressed in the AF duringdevelopment, we focused on Erg, a transcription factor that isknown to be involved in skeletal development [20–22]. Insclerotome cell cultures TGF-β is able to increase the expressionof Erg, along with other markers of the AF. The localization of Ergto the AF during development and its regulation by TGF-β led usto investigate its role in controlling differentiation of AF frommesenchymal progenitor cells. Our original hypothesis was that

Fig. 4 – ERG decreases cartilage gene expression and increasesSca1. Quantitative real-time RT PCR using RNA isolated fromsclerotome micromass cultures transduced with Ad-Erg orAd-GFP for 3 days. No effects of Erg over-expression on Fmod,Adamtsl2, or Scx mRNA levels were observed. Maf and Sox9mRNA were significantly decreased and Sca1 mRNA wassignificantly increased. RT-PCR data was analyzed by REST.Expression relative to Ad-GFP transduced cells is shown (set to1.0, dashed line). HPRT was used as the normalization control.Asterisks indicate p value, n¼13 for Maf, Sox9, and Sca1, n¼9for Fmod and Scx, and n¼8 for Adamtsl2. Fig. 5 – Erg acts synergistically with TGF-β to regulate specific

markers for mesenchymal cell differentiation. (A, B) Quantitativereal-time RT-PCR using RNA isolated from sclerotomemicromass cultures transduced with Ad-Erg or Ad-GFP for twodays and then left untreated or treated with 1 ng/ml TGF-β for24 h. Expression is shown relative to untreated Ad-GFPtransduced cells (set to 1.0, dashed line). TGF-β and Ergcooperated to up-regulate Adamtsl2 and Scx (A) and down-regulate c-Maf (B). RT-PCR data analyzed by REST. HPRT wasused as the normalization control. Asterisks (�) indicatespo0.05 relative to the Ad-GFP, untreated control. The step linesindicate po0.05 between the two samples connected; n¼4.

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Erg acts as a down-stream target of TGF-β in controlling AFdifferentiation.The effect of ectopic expression of Erg in sclerotome cells was

partially similar to that of TGF-β treatment in that both inhibitedformation of Alcian blue stained hyaline cartilage nodules. Inhibi-tion of Sox9 and c-Maf expression by Ad-Erg corresponded withinhibition of formation of chondrocytic nodules and indicatedthat the cells were not differentiating along the path of hyalinecartilage towards mineralization, which is the default for thesecultures. Nevertheless, none of the genes we analyzed as markersof AF or tendon differentiation were up-regulated in response toErg alone. This led us to consider other mesenchymal differentia-tion pathways including differentiation into adipocytes, smoothmuscle, or endothelial cells, which would have resulted inaccumulation of lipids within the cells, expression of αSMA, orPecam. Since Erg was previously shown to be involved in vasculardevelopment and global deletion of Erg results in early embryoniclethality due to severe hemorrhaging [37] it was important for usto exclude differentiation into either smooth muscle or endothe-lial cells. We did not observe changes in expression of any of theserelevant markers in Ad-Erg infected cells. Since these mesench-ymal derivatives were excluded, we were left with the possibilitythat the cells were remaining in an undifferentiated state.Erg expression resulted in increased levels of Sca1 mRNA, a

known marker for pluripotency in mouse [35], suggesting Erg mayhelp to maintain the cells in an undifferentiated state until they canrespond to the appropriate growth factors during development. Inhematopoietic cells, it has been shown that Erg is involved inmaintaining the stem cell population [38]. This suggested thepossibility that Erg is involved in maintaining a pool of undiffer-entiated cells in the sclerotome during development. The cellswould then be primed to receive an instructive signal from TGF-β

to drive differentiation to AF. Knowing that Erg like other ETS familymembers is capable of acting as a co-transcription factor, we lookedfor effects of Erg on TGF-β signaling in the sclerotome cells. Theresults indicated that Erg cooperates with TGF-β to enhance theexpression of Adamtsl2 and Scx mRNA. Although Fmod is a markerof the AF and is regulated by TGF-β, Erg did not affect expression ofFmod either alone or in combination with TGF-β. It is possible thatfor Fmod we were not looking at the right dose range of TGF-β tocooperate with Erg or, more likely, Erg only modulates expression ofa subset of TGF-β responsive genes. Cooperation between TGF-β andErg to regulate Adamtls2, Scx, and c-Maf raise the possibility that Ergcould interact directly with Smad3 to promote or repress transcrip-tion of specific genes. Previous reports indicating that Erg canstimulate a common TGF-β reporter element support this hypothesis[17]. Here we show through immunoprecipitation followed bywestern blot that Erg and Smad3 are present together in proteincomplexes in primary sclerotome cells. We did not test the interac-tion of Erg with other Smads including the BMP associated Smads1/5/8 so it is possible that Erg could interact with other Smads. Inaddition, at this time we do not know if or how TGF-βmodulates theinteraction between Erg and Smad3.

Fig. 6 – Erg and Smad3 interact in a protein complex. (A) Cellswere infected with Ad-GFP or Ad-Erg virus for 48 h. ControlRabbit IgG or Rabbit anti-Smad3 antibody was used forimmunoprecipitation. The immuno-complexes were subjectedto western blot analysis using a goat antibody to Erg. Highlevels of Erg were detected in Smad3 immuno-complexes fromAd-Erg infected cells relative to those from Ad-GFP infectedcells or from immuno-complexes from IgG precipitatedcontrols, which indicate some background signal likely fromthe IgG heavy chain. An immunoblot using a sample of theprotein lysates before immunoprecipitation (input) showsoverexpression of Erg in the Ad-Erg infected samples. (B)Endogenous Erg was immunoprecipitated from protein lysatesusing a goat anti-Erg antibody. The immuno-complexes wereseparated on gels for western blot analysis using a Rabbit anti-Smad3 antibody. Smad3 was detected in the Erg immuno-complexes. Western blot of samples of the protein lysatesbefore immunoprecipitation (input) show expressionof Smad3.

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Although Erg appears to block formation of hyaline cartilage insclerotome cultures it is not sufficient to induce differentiation ofmesenchymal cells on its own. This suggests that blocking thedefault chondrogenic pathway is not sufficient to induce differentia-tion along another lineage and an instructive signal, like TGF-β, isalso required. In addition to instructive signals that promote AFdifferentiation, an environment permissive for AF differentiation iscritical during IVD development. It is possible that Erg mediatedinhibition of chondrogenesis in the disc space provides this permis-sive environment. Early in development of the axial skeleton, BMP issynthesized by cells that will form the IVD. BMP protein is thentransported and concentrated in the area where the vertebral bodywill form [39,40]. To prevent chondrogenesis and subsequentendochondral bone formation in the IVD space, it is necessary toblock the action of BMP in this area. Multiple BMP inhbitors aresynthesized and localized to the developing IVD including Nogginand Chordin [8]. It has been shown in other model systems thatTGF-β can antagonize BMP signaling although the mechanism is notknown [41]. It is possible that Erg could act as an additional methodto prevent BMP mediated chondrogenesis in the IVD space.

In summary, Erg is expressed in the AF and its expression isregulated by TGF-β. Ectopic expression of Erg is not sufficient toinduce AF differentiation; however, TGF-β and Erg act synergis-tically to regulate Adamtsl2, a marker of AF. We propose that Ergmodulates TGF-β activity during development of the IVD.

Acknowledgments

This work was supported by a grant from the National Institutesof Health, R01AR053860, to RS. MKC was supported by the Dental

Academic Research Training (DART) grant from the NationalInstitute of Dental and Craniofacial Research, T32-DE176707/T90-DE022736.

r e f e r e n c e s

[1] B. Christ, R. Huang, M. Scaal, Formation and differentiation of theavian sclerotome, Anat. Embryol. (Berl) 208 (2004) 333–350.

[2] B. Christ, R. Huang, J. Wilting, The development of the avianvertebral column, Anat. Embryol. (Berl) 202 (2000) 179–194.

[3] M. Cox, R. Serra, Development of the intervertebral disc, in:I.M. Shapiro, M.V. Risbud (Eds.), The Intervertebral Disc, Springer,Vienna, 2014, pp. 33–51.

[4] Y. Saga, H. Takeda, The making of the somite: molecular events invertebrate segmentation, Nat. Rev. Genet. 2 (2001) 835–845.

[5] B. Christ, M. Scaal, Formation and differentiation of avian somitederivatives, Adv. Exp. Med. Biol. 638 (2008) 1–41.

[6] R. Huang, Q. Zhi, B. Brand-Saberi, B. Christ, New experimentalevidence for somite resegmentation, Anat. Embryol. (Berl) 202(2000) 195–200.

[7] R. Pelton, M. Dickinson, H. Moses, B. Hogan, In situ hybridizationanalysis of TGF-beta3 RNA expression during mouse develop-ment: comparative studies with TGF-beta1 and -beta2, Devel-opment 110 (1990) 600–620.

[8] I.B. Robertson, D.B. Rifkin, Unchaining the beast; insights fromstructural and evolutionary studies on TGFbeta secretion,sequestration, and activation, Cytokine Growth Factor Rev.. 24355–372.

[9] J. Wrana, L. Attisano, R. Wieser, F. Ventura, J. Massague,Mechanism of activation of the TGF-beta receptor, Nature 370(1994) 341–347.

[10] S. Shi, C. Ciurli, A. Cartman, I. Pidoux, A. Poole, Y. Zhang,Experimental immunity to the G1 domain of the proteoglycanversican induces spondylitis and sacroiliitis, of a kind seen inhuman spondylarthropathies, Arthritis Rheum. 48 (2003) 2903–2915.

[11] M. Baffi, M. Moran, R. Serra, Tgfbr2 regulates the maintenance ofboundaries in the axial skeleton, Dev. Biol. 296 (2006) 363–374.

[12] M. Baffi, E. Slattery, P. Sohn, H. Moses, A. Chytil, R. Serra,Conditional deletion of the TGF-beta type II receptor in Col2aexpressing cells results in defects in the axial skeleton withoutalterations in chondrocyte differentiation or embryonic devel-opment of long bones, Dev. Biol. 276 (2004) 124–142.

[13] P. Sohn, M. Cox, D. Chen, R. Serra, Molecular profiling of thedeveloping mouse axial skeleton: a role for Tgfbr2 in the devel-opment of the intervertebral disc, BMC, Dev. Biol. 10 (2010) 29.

[14] E.S. Reddy, V.N. Rao, T.S. Papas, The erg gene: a human generelated to the ets oncogene, Proc. Natl. Acad. Sci. U S A 84 (1987)6131–6135.

[15] A.D. Sharrocks, The ETS-domain transcription factor family, Nat.Rev. Mol. Cell Biol. 2 (2001) 827–837.

[16] A. Verger, M. Duterque-Coquillaud, When Ets transcriptionfactors meet their partners, Bioessays 24 (2002) 362–370.

[17] Y.H. Im, H.T. Kim, C. Lee, D. Poulin, S. Welford, P.H. Sorensen,C.T. Denny, S.J. Kim, EWS-FLI1, EWS-ERG, and EWS-ETV1 onco-proteins of Ewing tumor family all suppress transcription oftransforming growth factor beta type II receptor gene, CancerRes. 60 (2000) 1536–1540.

[18] Y. Ganan, D. Macias, M. Duterque-Coquillaud, M.A. Ros,J.M. Hurle, Role of TGF beta s and BMPs as signals controlling theposition of the digits and the areas of interdigital cell death in thedeveloping chick limb autopod, Development 122 (1996) 2349–2357.

[19] V. Vlaeminck-Guillem, S. Carrere, F. Dewitte, D. Stehelin,X. Desbiens, M. Duterque-Coquillaud, The Ets family member Erggene is expressed in mesodermal tissues and neural crests at

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 8 ( 2 0 1 4 ) 4 1 0 – 4 1 8418

fundamental steps during mouse embryogenesis, Mech. Dev. 91(2000) 331–335.

[20] M. Iwamoto, Y. Higuchi, E. Koyama, M. Enomoto-Iwamoto,K. Kurisu, H. Yeh, W.R. Abrams, J. Rosenbloom, M. Pacifici,Transcription factor ERG variants and functional diversification ofchondrocytes during limb long bone development, J. Cell Biol.150 (2000) 27–40.

[21] M. Iwamoto, Y. Higuchi, M. Enomoto-Iwamoto, K. Kurisu,E. Koyama, H. Yeh, J. Rosenbloom, M. Pacifici, The role of ERG (etsrelated gene) in cartilage development, Osteoarthr. Cartil. 9(Suppl A) (2001) S41–S47.

[22] M. Iwamoto, Y. Tamamura, E. Koyama, T. Komori, N. Takeshita,J. Williams, T. Nakamura, M. Enomoto-Iwamoto, M. Pacifici,Transcription factor ERG and joint and articular cartilage for-mation during mouse limb and spine skeletogenesis, Dev. Biol.305 (2007) 40–51.

[23] P. Dhordain, F. Dewitte, X. Desbiens, D. Stehelin, M. Duterque-Coquillaud, Mesodermal expression of the chicken erg geneassociated with precartilaginous condensation and cartilagedifferentiation, Mech. Dev. 50 (1995) 17–28.

[24] V. Hamburger, The stage series of the chick embryo, Dev. Dyn.195 (1992) 273–275.

[25] R.D. Riddle, R.L. Johnson, E. Laufer, C. Tabin, Sonic hedgehog mediatesthe polarizing activity of the ZPA, Cell 75 (1993) 1401–1416.

[26] A. Spandidos, X. Wang, H. Wang, S. Dragnev, T. Thurber, B. Seed,A comprehensive collection of experimentally validated primersfor polymerase chain reaction quantitation of murine transcriptabundance, BMC Genomics 9 (2008) 633.

[27] X. Wang, B. Seed, A. PCR primer bank for quantitative geneexpression analysis, Nucleic Acids Res. 31 (2003) e154.

[28] M.W. Pfaffl, G.W. Horgan, L. Dempfle, Relative expression soft-ware tool (REST) for group-wise comparison and statisticalanalysis of relative expression results in real-time PCR, NucleicAcids Res. 30 (2002) e36.

[29] C. Lorda-Diez, J. Montero, C. Martinez-Cue, J. Garcia-Porrero,J. Hurle, Transforming growth factors beta coordinate cartilageand tendon differentiation in the developing limb mesenchyme,J. Biol. Chem. 284 (2009) 29988–29996.

[30] H. Seo, R. Serra, Deletion of Tgfbr2 in Prx1-cre expressingmesenchyme results in defects in development of the long bonesand joints, Dev. Biol. 310 (2007) 304–316.

[31] C.I. Lorda-Diez, J.A. Montero, M.J. Diaz-Mendoza, J.A. Garcia-Porrero, J.M. Hurle, betaig-h3 potentiates the profibrogenic effect

of TGFbeta signaling on connective tissue progenitor cellsthrough the negative regulation of master chondrogenic genes,Tissue Eng. Part A 19 448–457.

[32] B. Yoon, D. Ovchinnikov, I. Yoshii, Y. Mishina, R. Behringer,K. Lyons, Bmpr1a and Bmpr1b have overlapping functions andare essential for chondrogenesis in vivo, Proc. Natl. Acad. Sci. U SA 102 (2005) 5062–5067.

[33] E. Berthet, C. Chen, K. Butcher, R.A. Schneider, T. Alliston,M. Amirtharajah, Smad3 binds scleraxis and mohawk andregulates tendon matrix organization, J. Orthop. Res. 31 (2013)1475–1483.

[34] W. Huang, N. Lu, H. Eberspaecher, B. De Crombrugghe, A newlong form of c-Maf cooperates with Sox9 to activate the type IIcollagen gene, J. Biol. Chem. 277 (2002) 50668–50675.

[35] S. Sun, Z. Guo, X. Xiao, B. Liu, X. Liu, P.H. Tang, N. Mao, Isolation ofmouse marrow mesenchymal progenitors by a novel and reliablemethod, Stem Cells 21 (2003) 527–535.

[36] Q. Zhao, H. Eberspaecher, V. Lefebvre, B. De Crombrugghe,Parallel expression of Sox9 and Col2a1 in cells undergoingchondrogenesis, Dev. Dyn. 209 (1997) 377–386.

[37] P. Vijayaraj, A. Le Bras, N. Mitchell, M. Kondo, S. Juliao,M. Wasserman, D. Beeler, K. Spokes, W.C. Aird, H.S. Baldwin,P. Oettgen, Erg is a crucial regulator of endocardial-mesenchymaltransformation during cardiac valve morphogenesis, Develop-ment 139 3973–3985.

[38] S. Taoudi, T. Bee, A. Hilton, K. Knezevic, J. Scott, T.A. Willson,C. Collin, T. Thomas, A.K. Voss, B.T. Kile, W.S. Alexander,J.E. Pimanda, D.J. Hilton, ERG dependence distinguishes devel-opmental control of hematopoietic stem cell maintenance fromhematopoietic specification, Genes Dev. 25 251–262.

[39] L. Zakin, C.A. Metzinger, E.Y. Chang, C. Coffinier, E.M. De Robertis,Development of the vertebral morphogenetic field in the mouse:interactions between crossveinless-2 and twisted gastrulation,Dev. Biol. 323 (2008) 6–18.

[40] L. Zakin, E.Y. Chang, J.L. Plouhinec, E.M. De Robertis,Crossveinless-2 is required for the relocalization of Chordinprotein within the vertebral field in mouse embryos, Dev. Biol.347 (2010) 204–215.

[41] T.F. Li, M. Darowish, M.J. Zuscik, D. Chen, E.M. Schwarz,R.N. Rosier, H. Drissi, R.J. O’Keefe, Smad3-deficient chondrocyteshave enhanced BMP signaling and accelerated differentiation, J.Bone Miner. Res. 21 (2006) 4–16.