Smad3IsaKeyNonredundantMediatorofTransforming … · Smad3IsaKeyNonredundantMediatorofTransforming GrowthFactorβ SignalinginNmeMouseMammary EpithelialCells JoannaDzwonek,1,2OlenaPreobrazhenska,1,2SilviaCazzola,1

Smad3 Is a Key Nonredundant Mediator of TransformingGrowth Factor β Signaling in Nme Mouse MammaryEpithelial Cells

Joanna Dzwonek,1,2 Olena Preobrazhenska,1,2 Silvia Cazzola,1,2 Andrea Conidi,1,2

Ann Schellens,1,2 Maarten van Dinther,3 Andrew Stubbs,4 Anke Klippel,5 Danny Huylebroeck,1,2

Peter ten Dijke,3 and Kristin Verschueren1,2

1Laboratory of Molecular Biology, Center for Human Genetics, K.U.Leuven; 2Department of Molecular andDevelopmental Genetics (VIB11), VIB, Leuven, Belgium; 3Molecular Cell Biology, Leiden University Medical Center,Leiden, the Netherlands; 4Department of Bioinformatics, Erasmus University Medical Center, Rotterdam, theNetherlands; and 5Silence Therapeutics, Berlin, Germany

AbstractSmad2 and Smad3 are intracellular mediators oftransforming growth factor β (TGFβ) signaling that sharevarious biochemical properties, but data emerging fromfunctional analyses in several cell types indicate that thesetwo Smad proteins may convey distinct cellular responses.Therefore, we have investigated the individual roles ofSmad2 and Smad3 in mediating the cytostatic andproapoptotic effects of TGFβ as well as their function inepithelial-to-mesenchymal transition. For this purpose, wetransiently depleted mouse mammary epithelial cells (Nme)of Smad2 and/or Smad3 mainly by a strategy relying onRNaseH-induced degradation of mRNA. The effect of suchdepletion on hallmark events of TGFβ-drivenepithelial-to-mesenchymal transition was analyzed,including dissolution of epithelial junctions, formation ofstress fibers and focal adhesions, activation ofmetalloproteinases, and transcriptional regulation ofacknowledged target genes. Furthermore, we investigatedthe effect of Smad2 and Smad3 knockdown on theTGFβ-regulated transcriptome by microarray analysis. Ourresults identify Smad3 as a key factor to triggerTGFβ-regulated events and ascribe tumor suppressor aswell as oncogenic activities to this protein. (Mol Cancer Res2009;7(8):1342–53)

IntroductionTransforming growth factor β (TGFβ) exerts multiple ef-

fects on several cell types and signals by binding to and acti-vating receptor complexes consisting of type I and type IIserine-threonine kinase receptors. These complexes trigger sev-eral intracellular transduction pathways, one of them relying onthe activation of Smad2 and Smad3. On direct phosphorylationby type I receptors, these Smads associate with Smad4. Thecomplexes then accumulate in the nucleus where they regulatetranscription through intricate low-affinity binding to DNA andinteraction with a wide variety of other transcriptional regula-tors (1, 2) often in a chromatin remodeling context (3).

In epithelial cells, TGFβ is known to act as tumor suppressorthrough its proapoptotic and cytostatic activities. TGFβ can alsoinduce epithelial-to-mesenchymal transition (EMT). EMT is adynamic process in which cells loose their epithelial phenotype,including their polarized character, mainly through the well-documented down-regulation of components of the differentcellular junctions. This weakens the homotypic adhesive forcesbetween cells and contributes as such to their increased inva-siveness. EMT is also accompanied by degradation of the base-ment membrane by matrix metalloproteinases (MMP), changesin the composition of the extracellular matrix, de novo expres-sion of several mesenchymal proteins, and alteration of thecytoskeleton (4-6). EMT has obtained wide attention as it isthought to be one of the underlying forces, at the cellular level,to drive progression of benign epithelial tumors into malignantinvasive and, ultimately, metastatic cancers. Recently, EMTwas also proposed to contribute to the generation of cells withproperties of cancer stem cells (7). There is ample experimentalevidence for the initial role of TGFβ as tumor suppressor as wellas subsequent promoter of carcinoma progression in vivo (8),and in vivo evidence for the existence of EMT in breast cancerwas obtained recently (9). The tumor-suppressive and oncogen-ic activities of TGFβ have been proposed to be mediated bydistinct pathways, with the suppressive pathway being inacti-vated in carcinomas and the oncogenic one not. Altogether,unraveling the signaling networks that govern the activities ofTGFβ in epithelial cells will remain crucial for the devel-opment of new strategies for future therapeutic interventionin cancer.

It is clear that besides Smads several other signaling com-ponents and modules are at play in TGFβ-induced EMT,

Received 12/8/08; revised 6/6/09; accepted 6/8/09; published OnlineFirst 8/11/09.Grant support: EpiPlastCarcinoma EU Marie Curie RTN Grant (K. Verschuerenand P. ten Dijke), VIB VIB7 and VIB11 (K. Verschueren and D. Huylebroeck),FEBS long-term fellowship (J. Dzwonek), and Dutch Cancer Society andBRECOSM EU project 503224 (P. ten Dijke).The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).Current addresses for O. Preobrazhenska: Department of Pathology and LaboratoryMedicine, University of British Columbia, Vancouver, British Columbia, Canada.Current addresses for A. Schellens: Laboratory of Developmental Genetics, VIB,Leuven, Belgium. Current addresses for A. Klippel: Wyeth Oncology Discovery,Pearl River, New York.Request for reprints: Kristin Verschueren, VIB Department of Molecular andDevelopmental Genetics (VIB11), Laboratory of Molecular Biology (Celgen),Campus Gasthuisberg K.U.Leuven, O&N1, Herestraat 49, Box 812, B-3000Leuven, Belgium. Phone: 32-16-34-59-16; Fax: 32-16-34-59-33. E-mail: [email protected] © 2009 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-08-0558

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including mitogen-activated protein kinases (10). It remains tobe determined what the precise contribution of each of the ac-knowledged TGFβ signaling components is in epithelial cells.Overexpression of a mutant type I receptor incapable of bindingand activating Smads but retaining the ability to activate mitogen-activated protein kinase signaling could not trigger the EMTresponse in normal breast epithelial cells (NMuMG; ref. 11),pointing at an essential role of Smad activation in EMT. The roleof the co-Smad Smad4 as mediator of the tumor-suppressive ac-tivities of TGFβ as well as EMT in mammary epithelial cells hasalready been investigated, and a role of Smad4 in metastasis ofbreast cancer cells in vivo has been shown (12). However, thecontribution of Smad2 and Smad3 to each of the distinct signal-ing events within epithelial cells has not been sufficiently ad-dressed and hence compared, and in some cases, such analysishas yielded conflicting results. For example, overexpression of adominant-negative form of Smad3 to levels that blocked growthinhibition and transcriptional responses to TGFβ was reportednot to inhibit EMT in mammary epithelial cells (13). In contrast,another study that used overexpression of dominant-negativeSmad2 or Smad3 attributed essential signaling functions to bothSmads in EMT within the same cells (13, 14). Overproduction ofthe mutant Smad proteins in these studies most likely blockedgeneral access of the endogenous Smad2 and Smad3 to activatedreceptor complexes in a nonspecific way and their subsequentbinding to Smad4, thus yielding little information about theindividual role(s) of Smad2 and Smad3 in TGFβ signaling inepithelial cells.

In this study, we carried out a comprehensive functionalanalysis of Smad2 and Smad3 in Nme cells; these cells consti-tute an acknowledged in vitro model system to study variousTGFβ activities. We mainly used a transient loss-of-functionapproach using GeneBlocs (GB), which are antisense oligonu-cleotides (AS-ON) of the third generation (15). These hybridDNA:RNA and 21 nucleotide–long AS-ONs hybridize to theirtarget mRNA and induce its degradation by RNaseH. This typeof modified AS-ONs has been shown to efficiently suppress/knock down expression of genes and has already been usedto develop AS-ON–based therapies in clinical trials for differ-ent diseases, including cancer (16-18).

ResultsSpecific Knockdown of Smad2 and Smad3 Using GBs

Cultured mouse mammary epithelial NMuMG cells are het-erogeneous with regard to E-cadherin distribution as cells withand without junctional E-cadherin are found. This obviouslycomplicates the interpretation of effects on cellular junctionscaused by transient Smad knockdown. Therefore, to facilitateour analysis, we used a subclone of NMuMG cells (Nme cells),which make up a homogeneous E-cadherin–producing cell pop-ulation. Similar to the parental cells, Nme cells have been docu-mented to undergo EMT on stimulation with TGFβ (14). In ourexperiments with Nme cells, the most discernible responses toTGFβ began to occur 48 to 72 hours after treatment, but thechanges in cell morphology could already be detected 24 hoursafter stimulation (data not shown). To carry out transient knock-down of Smads in Nme cells, modified single-stranded AS-ONs(called GBs) were used, which induce RNaseH-mediated degra-

dation of target Smad transcripts (15). As a negative control ineach experiment, cells were transfected with control oligonu-cleotides, which do not induce Smad mRNA degradation [mis-match (MM) control]. We first analyzed the dynamics of Smad2and Smad3 mRNA and protein depletion in TGFβ-stimulatedcells. TGFβ (TGFβ1) was added to cells 16 hours after transfec-tion and knockdown was analyzed in cells exposed to TGFβ forup to 48 hours. Levels of mRNA and protein were assessed byquantitative reverse transcription-PCR (q-RT-PCR) and Westernblotting, respectively, and compared with levels in MM-trans-fected cells (Fig. 1A and B). Our results show that knockdownat the steady-state Smad2 and Smad3 mRNA and protein levelspersisted up to at least 36 hours of TGFβ stimulation. After48 hours, the degree of knockdown started to wear off signifi-cantly and the levels of Smad mRNA and protein began to in-crease again (data not shown). Importantly, the degree ofrespective protein depletion induced by either Smad2 or Smad3GBs was consistent and comparable (Fig. 1B). Knockdown ofeach Smad by its matching GB was selective because attenuationof one Smad did not significantly affect expression of the otherSmad, with one exception: indeed, one of the GBs that was orig-inally designed to knock down Smad2 acted as a silencer ofboth Smad2 and Smad3 (and therefore is indicated here as2&3 GB; Fig. 1). Importantly, the level of Smad2 phosphoryla-tion following stimulation by TGFβ during 36 hours (Fig. 1C)or shorter (Supplementary Fig. S4) was not affected on knock-down of Smad3 and vice versa. Overall, in multiple experi-ments, knockdown using the GBs was found to be consistentand reproducible.

Depletion of Smad3 Interferes with TGFβ-InducedChanges in Cell Morphology, Cell Junctions, andCytoskeleton as well as with the Induction of MMPs

To obtain an overall view on the consequences of Smadknockdown, we first investigated TGFβ-induced changes incell morphology, dissolution/loss of epithelial junctions atcell-cell contacts, redistribution of filamentous actin (F-actin)from cell-cell contacts into stress fibers, and formation of focaladhesions, respectively. Knockdown of Smad3 abolished eachof these induced responses, whereas cells transfected with theMM control responded normally to TGFβ (Figs. 2 and 3).Strikingly, the Smad2 GB did not affect any of these inducedchanges and the response of the cells was comparable with theresponse of cells transfected with the MM control (Figs. 2A and3). Likewise, Smad2 depletion did not interfere with the abilityof TGFβ to down-regulate junction protein levels as analyzedby Western blotting, whereas Smad3 depletion did (Fig. 2B).Simultaneous knockdown of both Smad2 and Smad3, usingthe 2&3 GB (see above), did not cause visibly more drastic ef-fects than knockdown of Smad3 alone (Figs. 2 and 3). Analysisof the dynamics of changes in cell shape using time-lapse mi-croscopy showed a significant block in Smad3-depleted cells ofthe normal morphologic response. In contrast, Smad2 knock-down cells underwent TGFβ-induced changes in cell shape withdynamics comparable with these of MM control–transfectedcells (data not shown). Importantly, disruption of Smad2 orSmad3 function in nonstimulated cells did not cause any effecton cell morphology, epithelial junctions, and the cytoskeleton(Supplementary Figs. S1 and S2). To further substantiate our

Smad2 and Smad3 in TGFβ Signaling

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data obtained with the GBs, we also verified cell responses onSmad depletion by transient transfection of small interferingRNA (siRNA) directed against Smad2 and Smad3, and similarresults were obtained (Supplementary Fig. S3).

Another process associated with EMT is the increased abilityof cells to migrate and invade surrounding tissues in vivo andappropriate gel matrices in vitro, which requires the degradation

of extracellular matrix proteins by specific proteases. TGFβ isknown to induce MMP-2 and MMP-9 mRNA expression inNme cells (see also Figs. 4A and 7A and next section). Knock-down of Smad3 or Smad2&3 (using the 2&3 GB) abolished thisinduction (Fig. 4A), whereas depletion of Smad2 slightly affect-ed the level of induced MMP-2 (see Fig. 7A below). Analysis ofMMP-9 activity in conditioned medium from TGFβ-stimulated

FIGURE 1. Knockdown of Smads in Nme cells using GBs strongly and specifically inhibits Smad steady-state mRNA and protein levels. GBs efficientlydown-regulate Smad2 and Smad3 mRNA and protein expression in Nme cells. Cells were transfected with GBs specific for Smad2 (2 GB), Smad3 (3 GB),and Smad2 and Smad3 together (2&3 GB; see text) or with the MM control. After 16 h, the cells were treated with TGFβ (5 ng/mL) for 0, 6, and 36 h. RNA wasprepared and subjected to q-RT-PCR (SYBR Green). A. Smad mRNA levels are shown relative to the mRNA levels of glyceraldehyde-3-phosphate dehy-drogenase (GAPDH). Columns, mean of three repeats in each experiment; bars, SD. B. Extracts from Nme cells were analyzed by Western blotting todocument total Smad content using anti-Smad2 or anti-Smad3 antibody; α-tubulin protein levels were used as internal loading control. C. Knockdown ofone Smad does not affect activation by COOH-terminal phosphorylation (pSmad) of the other one. Nme cells were transfected with indicated GBs andstimulated for 36 h with TGFβ (5 ng/mL). Phosphorylation was assessed by Western blot analysis using anti–phospho-Smad2 or anti–phospho-Smad3antibody.

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cells by gelatin zymography confirmed these results (Fig. 4B).Strikingly, knockdown of Smad2 using the Smad2 GB caused aslightly enhanced induction by TGFβ of both MMP-9 genetranscription and gelatinase activity, but this was not confirmedby experiments using Smad2 siRNA (data not shown). This isin sharp contrast with consistent effects seen on cellular mor-phology responses following depletion of Smad2 and Smad3by either tool (GB or siRNA; Supplementary Fig. S3).

We were not able to examine MMP-2 activity in conditionedmedium because the serum used to grow the cells contained ahigh level of MMP-2 activity, and in addition, we were not ableto detect MMP-2 in conditioned medium from serum-starvedNme cells. Production and secretion of MMP proteins enablecells to degrade extracellular matrix proteins and become inva-sive. Despite several attempts, we also failed to examine therequirements of Smads in gaining cellular invasive propertiesin vitro. This is likely due to the multiple actions of TGFβon Nme cells. Indeed, its ability to induce EMT, together withthe cytostatic and proapoptotic activities, renders these cells un-suitable for this type of invasion analyses.

Nevertheless, our results obtained with Smad3 GB and con-firmed with siRNA clearly indicate that Smad3 is essential andthe major Smad is involved in triggering TGFβ-inducedresponses associated with EMT.

Overexpression of Human Smad3 Rescues the Effect ofEndogenous Smad3 Knockdown

Our data obtained from using GB and siRNA approachesindicate that Smad3 is the major Smad required to mediatethe studied responses to TGFβ. We wanted to verify by othermeans whether the effects caused by Smad3 GB transfectionwere specific. Therefore, we tried to rescue these defects byoverexpressing human Smad3 mRNA, which is not targetedfor degradation by the mouse-specific Smad3 GB. This wasdone by infecting the cells that are depleted for endogenousSmad3 with adenoviruses encoding Flag-tagged human Smad3.As a negative control, Smad3 knockdown cells were infectedwith adenoviruses carrying the gene encoding Cre recombinase(AdCre; Fig. 5). Infection with these recombinant adenovirusesinterfered neither with the ability of the cells to respond to

FIGURE 2. Smad3 but not Smad2 is required for TGFβ-induced changes in cell shape and repression of epithelial markers. After transfection with theindicated GB or MM control, Nme cells were treated or not with TGFβ (5 ng/mL) for 36 h and then analyzed by immunofluorescence (A) or Western blot (B)using anti–E-cadherin or anti–ZO-1 antibody. Bright-field pictures were also made to document change in cell shape. TGFβ-induced changes in cell mor-phology and the down-regulation and/or delocalization from cellular junctions of epithelial markers were blocked when Smad2 and Smad3 together (2&3 GB)or Smad3 alone was knocked down, whereas knockdown of Smad2 alone had no effect on change in cell shape and repression of epithelial markersE-cadherin and ZO-1.



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TGFβ nor with the efficiency of Smad3 knockdown. Thus,cells transfected with the Smad3 GB and infected with AdCredid not undergo EMT on stimulation with TGFβ (Fig. 5A,Sm3GB/AdCre) as shown by cell morphology and staining ofZO-1 and F-actin, whereas MM-transfected cells still did (Fig.5A, MM/AdCre). We did not investigate formation of focal ad-hesions because this was found to be induced by viral infectioneven in unstimulated cells (data not shown). Nevertheless, ad-enovirus-driven overexpression of human Smad3 clearly res-cued the defects in junction dissolution and stress fiberformation and in morphologic changes caused by depletionof endogenous Smad3 (Fig. 5A, Sm3GB/AdSm3) in ligand-stimulated cells. Likewise, the defects in MMP gene transcrip-tion and MMP activity in Smad3-depleted cells could berescued by exogenous Smad3 (Fig. 5B and C), even to someextent in unstimulated cells, due to overexpression of the ex-ogenous Smad3 (Fig. 5D). The ability of exogenous Smad3to rescue the defective responses to TGFβ observed follow-ing transfection with Smad3 GB indicated that the observeddefects were not caused by aspecific events.

Knockdown of Smad3 Interferes with the Cytostatic andProapoptotic Activities of TGFβ

Tumor-suppressive activities of TGFβ in epithelial cells relyon its ability to induce growth inhibition and apoptosis. Wehave studied the effect of Smad2 and Smad3 knockdown onthe cytostatic and proapoptotic activities of TGFβ, respectively.This was done by comparing bromodeoxyuridine (BrdUrd) in-corporation and the generation of cytoplasmic histone-associat-ed mononucleosomes and oligonucleosomes, respectively, inTGFβ-stimulated Nme cells transfected with the different GBs.Cells transfected with the MM control and treated with TGFβ for8 hours showed a statistically significant reduction in cell prolif-eration compared with unstimulated cells. Smad3 knockdown,unlike Smad2 knockdown, interfered with this antiproliferativeeffect (i.e., a statistically significant difference in proliferationof TGFβ-stimulated cells transfected with the Smad3 GB com-pared with the MM control was found; Fig. 6A). Likewise, de-pletion of Smad3 but not of Smad2 interfered with theproapoptotic activities of TGFβ, as detected after 24 hours of

stimulation with ligand (Fig. 6B; refer to Fig. 6C for depletedsteady-state levels of Smad2 and Smad3 mRNA levels in thisexperiment). This was further confirmed by comparison ofcleaved caspase-3 levels in several conditions after 36 hours ofTGFβ stimulation. Knockdown of Smad3 but not of Smad2 af-fected the TGFβ-dependent production of cleaved caspase-3.

q-RT-PCR and Gene Expression Profiling Data Confirmthe Role of Smad3 as a Critical Mediator of GeneResponses in TGFβ-Treated Nme Cells

To analyze the role of Smad2 and Smad3 as regulators ofgene transcription in response to TGFβ, we further analyzed

FIGURE 3. Induction of stress fiber and focal adhesion formation by TGFβ depends on Smad3. Following transfection with the indicated anti-Smad GB orMM control, cells were grown in the presence or absence of TGFβ (5 ng/mL for 36 h). Phalloidin staining to visualize F-actin and immunostaining using anti-paxillin antibody to visualize focal adhesions were done. TGFβ-induced stress fibers and focal adhesions in cells transfected with the MM control or Smad2GB were clearly visible, whereas knockdown of Smad3 or Smad2&3 completely abolished their formation.

FIGURE 4. TGFβ-mediated induction of MMP-9 expression is Smad3dependent. Following transfection with the Smad3 GB or the MM control,cells were grown in the presence or absence of TGFβ (5 ng/mL for 36 h).A. RNA was isolated and used for q-RT-PCR analysis with primers spe-cific to mouse MMP-9. MMP-9 mRNA was strongly induced from nearlyzero levels by TGFβ in cells transfected with the MM control. Knockdownby Smad3 or Smad2&3 GB diminished this induction. B. Gelatin zymogra-phy from conditioned medium from the cells used in A. TGFβ-inducedactivity (bottom) of MMP-9 was diminished in cells transfected with Smad3or Smad2&3 GB.

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the effect of their knockdown on the expression of acknowl-edged target genes for TGFβ. Induction of fibronectin-1mRNA and protein on stimulation of the Nme cells with TGFβwas abolished in cells depleted for any of the Smads analyzed,albeit to a different degree. Smad3 knockdown, and its com-bined knockdown with Smad2, had the strongest negative ef-fect on fibronectin-1 induction (Fig. 7A and B), whereasSmad2 knockdown only weakly affected this induced level.

Induction of TIMP-1, PAI-1, Lef-1, MMP-2, and ALK-5 mRNAby TGFβ was diminished in Smad3-depleted but not, or less so,in Smad2-depleted cells (Fig. 7A). Likewise, TGFβ-dependentrepression of Id2 transcription was reduced when Smad3 (butnot Smad2) production was attenuated by GB transfection.Importantly, the basal level of expression of these genesin unstimulated cells was not significantly affected by Smadknockdown.

FIGURE 5. The effect of Smad3 knockdown can be rescued by expression of a virus-transduced human Smad3 cDNA. A. Nme cells were transfectedwith Smad3 GB or MM control and, 5 h later, infected with adenoviruses encoding Cre recombinase (AdCre) or Flag-tagged human Smad3 (AdSm3). Twelvehours after infection, the cells were treated with TGFβ for the next 36 h. Immunocytochemistry using anti–ZO-1 or anti-Flag antibodies (red) and phalloidin(green) and 4′,6-diamidino-2-phenylindole (Dapi; blue) stainings were done. Bright-field photographs were taken to visualize cell morphology. B. mRNA wasprepared and subjected to real-time PCR using specific primers for mouse MMP-2 and MMP-9. C. Conditioned medium from these cells was analyzed bygelatin zymography. Defects caused by Smad3 depletion in cells transfected with Smad3 GB were rescued by synthesis of adenovirus-encoded humanSmad3 (Sm3GB/AdSm3). D. Level of exogenous adenovirus-driven expression of human Smad3 compared with endogenous Smad3.



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The results from the analysis of these transcriptional re-sponses prompted us to extend this analysis by gene expressionprofiling. Doing so, we also aimed to broaden our search forSmad2-dependent responses to TGFβ in these Nme cells. Weanalyzed gene responses after 6 hours of stimulation with TGFβto investigate early transcriptional events that are diminished orabolished by Smad2 or Smad3 knockdown. Quintuplicate sam-ples of Nme cells transfected with the MM control, the Smad2-and the Smad3-specific GBs, respectively, were stimulated withTGFβ (or not) for these 6 hours. Two of these samples were usedto confirm the efficacy of knockdown by Western blot and q-RT-PCR analysis (refer to Supplementary Fig. S4), whereas theremaining samples were used to compare gene expression pro-files in all these different conditions using mouse whole-genomeGeneChips (Affymetrix). The knockdown efficiency was com-parable for Smad2 and Smad3 in this experiment (refer to Sup-plementary Fig. S4). Overall, the microarray data yielded lists ofTGFβ-responsive genes and genes for which these responsesseemed dependent on Smad3 and only a very few on Smad2.We also identified genes, the regulation of which seemed Smad

independent (microarray data submitted at ArrayExpress, acces-sion number E-MEXP-1342). Verification of the response of aselected set of genes in an independent experiment by q-RT-PCR revealed that only the data sets with P values of ≤0.001could be validated. We therefore applied this stringent selectioncriterion to further define the genes whose expression signifi-cantly varied among the conditions analyzed. More specifically,we searched for TGFβ-responsive genes by comparing gene ex-pression profiles in unstimulated versus stimulated cells trans-fected with the MM control (i.e., genes differently expressedin MMSvsU, group A in Supplementary Table S1; refer to thelegend of this table for an explanation of the abbreviations usedto denominate the different groups). A total of 217 TGFβ-responsive transcripts were identified (118 up-regulated and 99down-regulated by TGFβ). We went on to identify in our datasets genes that were differentially expressed following TGFβstimulation of Smad3 GB–transfected cells (SM3GBSvsU,group B in Supplementary Table S1). In addition, we comparedgene expression profiles in TGFβ-stimulated cells transfectedwith the MM control versus the Smad3 GB. Such genes are listed

FIGURE 6. Smad3 knockdown affects cell proliferation and apoptosis in TGFβ-stimulated Nme cells. Nme cells were transfected with GBs for Smad2 andSmad3 and with MM control. The cells were treated (or not) with TGFβ for 8 or 24 h and then analyzed for their proliferation (A) and apoptosis (B). Columns,mean of three (cell proliferation assay) or four (apoptosis assay) repeats; bars, SD. The statistical significance of the difference between conditions wascalculated using the two-sided t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. A. Smad2 GB–transfected and MM control–transfected Nme cells are still subjectto the TGFβ-induced antiproliferative effect, whereas Smad3 GB–treated cells are not. B. Knockdown of Smad3 but not Smad2 blocked TGFβ-inducedapoptosis. C. Corresponding levels of Smad2 and Smad3 knockdown (mRNA) in cells used for the apoptosis assay. D. Knockdown of Smad3, but notof Smad2, inhibits production of cleaved caspase-3.

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as group C in Supplementary Table S1 (differentially expressedin SM3GBSvsMMS). Genes in groups D, F, and H representoverlaps between groups A, B, and C, and these embodyTGFβ-responsive genes that are dependent or independent onSmad3 for their response to TGFβ. More specifically, genesin group D are TGFβ responsive, and their response to theligand is partially affected on Smad3 knockdown (and hencethese genes are listed in group A as well as groups B and C).These genes are Snai1, Serpine 1 (PAI-1), and an unknown gene(2310007B03Rik). In addition, the 11 Affymetrix IDs in group Frepresent an overlap between group A and group C (Supplemen-tary Table S1). These IDs represent genes that have a nearly com-pletely abolished response to TGFβ on Smad3 knockdown.Indeed, expression of these TGFβ-sensitive genes is differentialin TGFβ-regulated cells transfected with the Smad3 GB versusthe MM control (compare SM3GBSvsMMS). Moreover, thesegenes lost response to TGFβ in Smad3 knockdown cells (compareSM3GBSvsU). Four of those Affymetrix IDs represent two genes:Id2 and Sdpr. Thus, this analysis yielded according to our stringentselection a total of nine TGFβ-sensitive and Smad3-dependentgenes. This set of disturbed responses caused by Smad3 knockdown(except for Sdpr) was confirmed by q-RT-PCR (see Table 1) in anindependent experiment. Examples of genes identified as beingregulated by TGFβ only in the presence of normal Smad3 levelsare Il-11, CD40, Id2, and Id3 (see Table 1). Altogether, genesin both of groups D and F are listed in the upper part of Table 1as genes that require Smad3 for TGFβ responsiveness.

Group H represents 24 other genes that retained full respon-siveness to TGFβ in Nme cells even on depletion of Smad3.More specifically, they are found in group A and are therefore

TGFβ sensitive as well as group B and are therefore still reg-ulated by TGFβ on knockdown of Smad3. Therefore, the reg-ulation of their expression by the ligand is Smad3 independent.Examples are Cxcl1, Keratin-20, Gjb3, and Sema4f (see lowerpart of Table 1).

We carried out a similar analysis comparing gene expressionprofiles in cells transfected with the MM control versus Smad2GB. This analysis yielded only six genes of which responses toTGFβ seemed to be dependent on Smad2. However, this de-pendence could not be confirmed in independent experiments,except for Snail. The transcriptional induction of the Snail genewas indeed affected by Smad2 knockdown, with levels reducedto 60% compared with the level in control cells, whereasknockdown of Smad3 caused a reduction to 40% (data notshown). Finally, further verification of transcript levels of theidentified Smad3-dependent genes by q-RT-PCR showed thatthe response of these to TGFβ did not require Smad2 whatso-ever. Altogether, and in sharp contrast with the results obtainedfor Smad3, the combination of microarray analysis and the ap-plication of stringent selection criteria did not yield a singlegene in Nme cells that significantly (except for Snail) or exclu-sively depended on Smad2 for its response to TGFβ.

DiscussionIn this study, we have transiently depleted Smad2 and/or

Smad3 from normal mammary epithelial (Nme) cells to analyzewhich aspects of TGFβ signaling in these depend on intactSmad2 or Smad3 activity. Several studies in different cell typeshave already aimed at distinguishing between the specific roles

FIGURE 7. Differential regulation of TGFβ-responsive genes by Smads. Following transfection with GB or the MM control, cells were grown in thepresence or absence of TGFβ (5 ng/mL for 36 h). mRNA (A) or protein (B) was isolated and analyzed by q-RT-PCR or Western blotting, respectively.Columns, mean of three repeats from each experiment; bars, SD. The knockdowns of all investigated Smads resulted in diminished induction of fibronectin-1mRNA (A) and protein (B) expression under TGFβ stimulation. The induction of TIMP-1, PAI-1, Lef-1, ALK-5, and MMP-2 as well as the repression of Id2mRNA expression by TGFβ were significantly abolished when normal Smad3 or Smad2&3 levels were abolished but not, or less so, when Smad2 wasknocked down.



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of Smad2 and Smad3, which share biochemical properties, inTGFβ signaling. The majority of these data were obtained fromthe assessment of transcriptional responses in Smad2 andSmad3 knockout cells, including fibroblasts, hepatocytes, aswell as epithelial cells from lens and renal tubules (19-24).Both of these latter types of epithelial cell also undergo EMTin response to TGFβ, which has been proposed to contribute tofibrosis in the kidney or in the lens after injury. In accordancewith our results obtained in the Nme cells used here, the datacollected from these other cell types point to Smad3 as an es-sential mediator of responses to TGFβ. Three reports fromstudies in human epithelial cell lines, including keratinocytesor hepatocytes, showed that Smad3 also has a prominent rolein TGFβ-mediated cell cycle arrest and apoptosis (25-27).

To the best of our knowledge, the data presented here pro-vide the first comprehensive and comparative analysis ofSmad2 and Smad3 function in hallmark steps of TGFβ-drivenEMT, transcriptional responses, and cytostatic and proapoptoticactivities, and this is within a single cell type. Moreover, we

used a transient knockdown strategy, which provides an unbi-ased approach to analyze Smad function without putative selec-tion for compensatory effects, which most likely occur inknockout cells. This is indeed important because the Smadknockout cells used in some studies (such as knockout fibro-blasts) can display disturbances in the basal levels of variousresponses when compared with wild-type cells (20). We havedone this transient knockdown by using novel tools (GBs). Thisantisense RNA:DNA hybrid oligonucleotides recruit RNaseHto the Smad2 or Smad3 target mRNA, causing its degradation.Several lines of evidence have indicated already that this RNa-seH-mediated antisense strategy is a valid and alternative ap-proach for siRNA-mediated depletion of gene products (15,25). Moreover, we have validated some of our results obtainedwith GBs also with the siRNA-based approach and the sameresults were obtained using either tool, except for the effectof Smad2 depletion on MMP-9 gene induction by TGFβ. Cellstransfected with the Smad2 GB displayed an enhanced TGFβ-dependent induction of the MMP-9 gene, but this was not

Table 1. Smad3 Dependence of TGFβ Responses in Nme Cells

Affymetrix ID Probe Name Gene Name TGFβ Response Smad3 Dependence

Microarray q-RT-PCR Microarray q-RT-PCR

1416239_at Ass1 Argininosuccinate synthetase 1 ↑ +1439221_s_at Cd40 B-cell surface antigen CD40 ↑ ↑ + +1422537_a_at Id2 Inhibitor of differentiation 2 ↓ ↓ + +1435176_a_at1416630_at Id3 Inhibitor of differentiation 3 ↓ +1449982_at Il11 Interleukin-11 ↑ ↑ + +1419534_at Olr1 Oxidized low-density lipoprotein receptor 1 ↑ ↑ + +1456888_at Pfkfb4 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 ↑ +1416778_at Sdpr Serum deprivation response ↓ ↓ + nc1443832_s_at1419149_at Serpine1 Plasminogen activator inhibitor 1 ↑ ↑ + +1448742_at Snai1 Snail ↑ ↑ + +1453511_at 2310007B03Rik ↑ +1429185_at 4631416L12Rik ↓ +1416786_at Acvr1 Activin A receptor, type I ↑ −1426719_at Apbb2 Amyloid β (A4) precursor protein binding, family B,

member 2 (Fe65-like)↑ −

1451340_at Arid5a AT-rich interactive domain 5A (MRF1-like) ↑ −1418025_at Bhlhb2 Basic helix-loop-helix domain containing, class B, 2 ↑ −1449300_at Cttnbp2nl CTTNBP2 NH2-terminal like ↑ −1457644_s_at Cxcl1 Chemokine (C-X-C motif) ligand 1 (melanoma

growth-stimulating activity, α)↓ −

1442434_at D8Ertd82e Homologue of rat pragma of Rnd2 ↑ −1424229_at Dyrk3 Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3 ↑ −1423100_at Fos v-fos FBJ murine osteosarcoma viral oncogene homologue ↑ −4551959_s_at Gclc Glutamate-cysteine ligase, catalytic subunit ↓ −1416715_at Gjb3 Gap junction protein, β3, 31 kDa ↑ −1440559_at Hmga2-ps1 High-mobility group AT-hook2, pseudogene 1 ↑ −1432478_a_at Ibrdc3 IBR domain containing 3 ↑ −1416200_at Il33 Interleukin-33 ↓ −1426284_at Krt20 Keratin 20 ↑ −1424596_s_at Lmcd1 LIM and cysteine-rich domains 1 ↑ −1417275_at Mal Mal, T-cell differentiation protein ↑ −1420461_at Mst1r Macrophage-stimulating 1 receptor (c-met–related tyrosine kinase) ↑ −1451527_at Pcolce2 Procollagen C-endopeptidase enhancer 2 ↑ −1450413_at Pgfbd Platelet-derived growth factor β polypeptide [simian sarcoma viral (v-sis)

oncogene homologue]↑ −

1449424_at Plek2 Pleckstrin 2 ↑ −1439768_x_at Sema4f Semaphorin 4F ↑ −1460197_a_at Steap4 Six-transmembrane epithelial antigen of the prostate family member 4 ↑ −1451450_at 1424694_at 2010011l20Rik ↑ −

NOTE: List of genes regulated by TGFβ [up-regulated (↑) or down-regulated (↓)] after 6 h of stimulation in a Smad3-dependent (+) and Smad3-independent (−) manner.Genes were scored as Smad3-dependent (+) or Smad3-independent (−) TGFβ targets as described in Materials and Methods. For most Smad3-dependent genes, theirregulation has been confirmed by q-RT-PCR as indicated as (+) in q-RT-PCR column. Genes in bold: tumor-related genes; genes in italics: EMT-related genes.Abbreviation: nc, not confirmed.

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confirmed using Smad2 siRNA. The discrepancy between theresults using both knockdown tools is not clear, but it maymainly emphasize the need to carry out knockdown studiesusing different tools.

The function of Smad3 as major mediator of TGFβ was fur-ther confirmed in our gene expression profiling experiments. Inthese, we identified 12 unique genes for which the responses toTGFβ were Smad3 dependent and 24 genes for which the re-sponse was Smad3 independent. A substantial fraction of thesegenes has already been functionally related to cancer [printed asbold in Table 1; i.e., as tumor-suppressing factors (such as Id2,Id3, and Gclc, which are down-regulated by TGFβ in this mod-el system) or as factors involved in metastasis (such as Cxcl1,Snail, and Il-11, which are induced)]. The list of Smad3-dependent genes is enriched for genes known to be involvedin EMT (i.e., 6 of 12 genes encode acknowledged players in thisprocess). In contrast, among the 24 Smad3-independent tran-scripts identified, only one gene has, thus far, been functionallyimplicated in EMT (Mst1r; ref. 28). Expression of this gene,which encodes a receptor tyrosine kinase, is induced by TGFβ.Activation of this kinase activity has been reported to attenuateTGFβ-induced apoptosis and promote phenotypic changes inepithelial cells (28). It will be of interest to determine which othersignaling components, such as Smad4, are implicated in the tran-scriptional regulation of Smad3-independent genes.

In our studies, next to the lack of detectable defect on most ofthe TGFβ-regulated cellular responses, we could find a few tar-get genes that responded differently to TGFβ in the absence ofnormal Smad2 levels. Indeed, the transcriptional induction ofSnail, MMP-2, fibronectin-1, PAI-1, and LEF-1 genes was foundto be attenuated. Overall, this effect was, however, not sufficientto detectably interfere with the EMT response in the Smad2-depleted cells. Altogether, we did not identify a single genefor which the response to TGFβ exclusively relied on Smad2.

Previous studies report Smad2 as attenuator of TGFβ signal-ing. For example, Smad2 knockout fibroblasts lacking Smad2were reported to be hyperresponsive to TGFβ (21). Anotherstudy showed that Smad2 knockout hepatocytes acquire mesen-chymal features and increased cell proliferation even in the ab-sence of exogenous TGFβ stimulation (29), suggesting thatSmad2 inhibits epithelial characteristics and proliferation, atleast of hepatocytes. Likewise, Smad2 knockout keratinocyteshave been reported to exhibit reduced levels of E-cadherin pro-tein and increased levels of nuclear Snail protein in vivo, thuscontributing to the accelerated formation and malignant pro-gression of chemically induced skin tumors in mice (30). More-over, depletion of Smad2 in human epithelial cells using siRNAwas reported to enhance the Smad3-dependent cytostatic re-sponse to TGFβ (26). In this study, we did neither find evi-dence for the role of Smad2 as a general attenuator of TGFβresponses in Nme cells nor discern an effect of Smad2 knock-down on epithelial markers in unstimulated cells.

Overall, our data indicate that activation of Smad3 constitu-tes a main trigger to initiate the EMT process, whereas normalSmad2 levels seemed not to be critical for EMT, at least in Nmecells and in the conditions and time window analyzed here. Ourdata also indicate that depletion of Smad3 but not of Smad2interfered with TGFβ-induced proapoptotic and antiprolifera-tive activities, thus further contributing to the emerging view

that Smad3 is the main mediator of these TGFβ-controlled pro-cesses. At this stage, we cannot totally exclude that the degreeof Smad2 knockdown obtained in our experiments, albeit con-sistent and substantial, is insufficient to interfere with normalSmad2 activity. This would imply that significant disturbancesin Smad2 protein levels somehow do not cause a Smad2 loss offunction. Alternatively, it is also possible that Smad3 can com-pensate for the loss of Smad2 in cells, whereas Smad2 cannotcompensate for the loss of Smad3. This would be in accordanceto our findings that double knockdown of Smad2 and Smad3causes the same defects as knockdown of Smad3 alone. As athird explanation, which is also in line with some of our data,Smad2 would rather regulate the amplitude of gene responsesto TGFβ. The sensitivity of the microarray analysis, especiallyin the context of small effects on transcriptional responsescaused by knockdown, might have precluded further definitionof Smad2-dependent processes in this study.

Taking all the obtained results together, we have shown thatSmad3 acts as a main mediator of the antiproliferative, proa-poptotic, and EMT responses to TGFβ, respectively, in non-transformed mammary epithelial cells using Nme cells as amodel. Therefore, within a single cell type, Smad3 may actas a mediator of both the tumor-suppressive as well as the in-vasion-promoting activity of TGFβ. A recent study reports thatin Ras-transformed malignant cells, the switch between bothactivities of TGFβ could be regulated by domain-specific phos-phorylation of Smad3. Indeed, in such transformed cells, c-JunNH2-terminal kinase was shown to constitutively phosphory-late the linker region of Smad2 and Smad3, which then trans-locate to the nucleus, resulting in transcription of genesinvolved in invasion and cell migration (31). For Smad3, thisphosphorylation by c-Jun NH2-terminal kinase simultaneouslyhas been proposed to block its COOH-terminal phosphoryla-tion by the activated receptor complex, concomitant with aber-rant expression of c-Myc and resistance to the growthsuppression in response to TGFβ. Such a mechanism could ex-plain the loss of Smad3-dependent cytostatic activity of TGFβin Ras-transformed malignant cells while retaining the Smad3proinvasive activities (31). Further studies of this type will berequired to investigate the contribution of Smad2 and/or Smad3in breast malignancies in vivo.

Materials and MethodsCell Line

Nme cells, an acknowledged subclone of NMuMG (32),were grown in DMEM supplemented with 10% fetal bovineserum, 2 mmol/L glutamine, and 10 μg/mL insulin.

Transfection with GB AS-ONsThe anti-Smad GBs and their corresponding MM control oli-

gonucleotides (Atugen) were used as previously described (25)and the GBs used for this study contained the following se-quences: Smad2 GB (no. 73263), 5′-aacccuGGTTGACAGacugag-3′;Smad2&3 GB (no. 73261), 5′-aacccuGGTTGACAGacugag-3′;Smad3 GB (no. 73294), 5′-uguaggcCATCCAGTTugacagg-3′;and MM GB (no. 22285), 5′-cuccaucTTCACTCAGguagcca-3′.

Transfections were carried out as follows: 300 nmol/L ofGB dilution in serum-free medium were mixed with 12 μg/mLof lipid (Argfectin-50, Atugen) and incubated at 37°C for



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20 min. Then, Nme cells in suspension (125,000/mL) wereadded directly to the GB-lipid complex. Final concentrationof GB was 30 nmol/L and 1.2 μg/mL of the lipid. The mediumwas replaced 16 h later with fresh medium containing TGFβ1(R&D Systems) at 5 ng/mL or with vehicle.

To knock down Smad2 and Smad3 by siRNA, ON-TAR-GET plus SMARTpool L-040707-00-005 and L-040706-00-0005 and, as control, the ON-TARGET plus Non-targeting pool(Dharmacon) were used. Cells were transfected in suspension(125,000/mL) using Lipofectamine 2000 (Invitrogen) accord-ing to the described protocol in 12-well plates. For each well,1 μL siRNA was added to 50 μL Opti-MEM and this mix wasadded to 100 μL Opti-MEM preincubated with 2 μLLipofectamine 2000 and incubated for 20 min at 37°C. Cellsuspension (300 μL) was subsequently added and the mixtureswere plated. An additional 550 μL of medium was added after5 h. The medium was replaced 24 h later with fresh mediumcontaining TGFβ1 (R&D Systems) at 5 ng/mL or with vehicle.

Cell Proliferation AssayNme cells (50 × 103) were transfected in suspension and plat-

ed for each condition in triplicate in wells of Black 96-well plateswith clear flat bottom (ViewPlate F TC, Perkin-Elmer). Sixteenhours later, the medium has been replaced with fresh mediumwith or without 5 ng/ml TGFβ1 for 6 h. After 6 h, the cells werelabeled for 2 h with BrdUrd in medium with or without TGFβ1.The plates were dried at 60°C for 1 h according to the kit proto-col (Cell Proliferation ELISA BrdUrd chemiluminescent, RocheApplied Science). BrdUrd incorporation has been detected bychemiluminescent reaction using a standard ELISA reader andis represented by arbitrary units on Fig. 6. The statistical signif-icance of the difference in BrdUrd incorporation between condi-tions was calculated using the two-sided t test.

Detection of ApoptosisApoptosis was analyzed by quantitative assessment of the

presence of cytoplasmic histone-associated mononucleosomesand oligonucleosomes using the Cell Death Detection ELISAkit (Roche Diagnostics). Cells were transfected in suspensionand plated for each condition in quadruplicate at a concentra-tion of 125,000 per well in 96-well plates. Sixteen hours later,the medium was replaced and cells were incubated with orwithout TGFβ1 for 24 h. Cells were then harvested and lysed.The Cell Death Detection ELISA was done according to thekit's protocol. The ELISA values were normalized to total cellnumbers per well (represented by arbitrary units in Fig. 6). Thestatistical significance of the difference in apoptosis betweenconditions was calculated using the two-sided t test.

Adenoviral InfectionAdenoviruses expressing the control protein, Cre recombi-

nase (AdCre), or Flag-tagged human Smad3 (AdSm3; ref.14) were used for the experiments. Nme cells were first trans-fected with GBs or the MM control. Five hours later, cells wereinfected with adenoviruses (multiplicity of infection, 200) for12 h and then medium was replaced with fresh medium con-taining vehicle or TGFβ1 at 5 ng/mL. Cells were assayed36 h after treatment with ligand.

Western BlottingCells were lysed in buffer containing 50 mmol/L Tris (pH 8),

150 mmol/L NaCl, 1% deoxycholic acid, 1% NP40, 0.1% SDS,1 mmol/L phenylmethylsulfonyl fluoride, and complete prote-ase inhibitor cocktail tablets (1 tablet for 25 mL of buffer;Roche). Protein concentration was measured using bicinchoni-nic acid protein assay kit (Pierce) and 10 μg of total proteinwere loaded on SDS-PAGE in 6% or 8% (w/v) Bis-Tris–containing polyacrylamide gels under reducing conditions.Proteins were then transferred to polyvinylidene difluoridemembrane. Blots were blocked with 5% fat-free milk inTBS-T (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% Tween)and then incubated with primary antibodies (1:1,000 in TBS-Twith 5% bovine serum albumin) at 4°C overnight to detectSmad2 (Transduction Laboratories), Smad3 (Zymed), phospho-Smad2 (Upstate), phospho-Smad3 (Acris), E-cadherin (Trans-duction Laboratories), ZO-1 (Chemicon), fibronectin (Sigma),α-tubulin (ProBio), and cleaved caspase-3. Next, the blots wereincubated with horseradish peroxidase–labeled secondary anti-body for 1 h at 24°C and then developed with enhanced chemi-luminescence Western blotting detection system (Amersham).Visualization was with Fuji photographic films. Blots werestripped and reprobed with α-tubulin as a loading control.

Gelatin ZymographyNme cells were transfected with the Smads or the MM con-

trol. After 16 h, the medium was changed and TGFβ1 or vehi-cle was added to the cells. After 36 h, cells were harvested andconditioned medium was collected and separated by electro-phoresis in an 8% polyacrylamide gel containing 1 mg/mL gel-atin as substrate. The amount of conditioned medium loadedper well was adjusted according to the protein concentrationof the cell lysates. After electrophoresis, gels were washedtwice for 10 min in 2.5% Triton X-100 and incubated overtwo nights in a reaction buffer [50 mmol/L Tris (pH 7.6), 10mmol/L CaCl2, 1 μmol/L ZnCl2, 1% Triton X-100, 0.02% so-dium azide] and stained with Brilliant Blue G-Colloidal Con-centrate (Sigma).

ImmunocytochemistryImmunofluorescence staining was done as described before

(33). Antibodies against E-cadherin, ZO-1, paxillin (Transduc-tion Laboratories), and Alexa Fluor phalloidin (MolecularProbes) to stain F-actin were used in 1:500 dilutions.

Real-time PCRRNA was extracted using the RNeasy Mini kit (Qiagen). Re-

verse transcription of 1 μg RNA was carried out by using Re-vertAid H Minus M-MuLV reverse transcriptase (Fermentas).Real-time PCR was done with SYBR Green PCR master mix(Applied Biosystems). The monitoring and quantitative analy-sis of PCR products were done with an ABI Prism 7000 Se-quence Detection System (Applied Biosystems). The amountof PCR product derived from each mRNA was normalized tothat from glyceraldehyde-3-phosphate dehydrogenase in thesame sample. Obtained values are represented as arbitrary unitson the graphs. All primers were design using Primer Expresssoftware (Applied Biosystems).

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cDNA Microarray AnalysisThe cDNA microarray analyses were done with RNAs iso-

lated from Nme cells after transfection with control GB: MM-,Smad3-, or Smad2-specific GBs (SM3GB and SM2GB). Six-teen hours after transfection, cells were treated or not with 5 ng/mLTGFβ1 for 6 h. Five independent repeats of the experimentwere carried out. Two of these were used to confirm the effi-ciency of the Smad2 and Smad3 knockdown by q-RT-PCR andWestern blot and three were labeled and hybridized with Gen-eChip Mouse Genome 430 2.0 Array (Affymetrix) according tothe manufacturer's instructions. This enables the analysis of theexpression of ∼45,000 probe sets corresponding to over 39,000transcripts. Raw and normalized data and more detailed proto-cols are available at the ArrayExpress public database (acces-sion number E-MEXP-1342).

Affymetrix scanner and the resultant .cel files were used insubsequent analysis workflow to identify differentially ex-pressed genes. Scanned images were first inspected for qualitycontrol using a variety of quality control tools from the Biocon-ductor open source environment for statistical analysis. Qualitycontrol consisted of visual examination of probe array images,scatter plots from replicates, hierarchical clustering and corre-lation plots of array hybridizations, RNA degradation plots, andnormalized unscaled SE plots (34). Feature intensity valuesfrom scanned arrays were normalized and reduced to expres-sion summaries using the Robust Multiarray Algorithm (35).Differentially expressed genes were determined using Bayesianadjusted t statistics from the linear models for microarray data(limma) corrected for multiple testing based on the false dis-covery rate (36, 37).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

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2009;7:1342-1353. Published OnlineFirst August 20, 2009.Mol Cancer Res Joanna Dzwonek, Olena Preobrazhenska, Silvia Cazzola, et al. Epithelial Cells

Signaling in Nme Mouse MammaryβGrowth Factor Smad3 Is a Key Nonredundant Mediator of Transforming

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Smad3IsaKeyNonredundantMediatorofTransforming … · Smad3IsaKeyNonredundantMediatorofTransforming GrowthFactorβ SignalinginNmeMouseMammary EpithelialCells JoannaDzwonek,1,2OlenaPreobrazhenska,1,2SilviaCazzola,1