MicroRNA-208ModulatesBMP-2-stimulatedMouse ... · MicroRNA-208ModulatesBMP-2-stimulatedMouse...

MicroRNA-208 Modulates BMP-2-stimulated MousePreosteoblast Differentiation by Directly Targeting V-etsErythroblastosis Virus E26 Oncogene Homolog 1*

Received for publication, January 19, 2010, and in revised form, June 21, 2010 Published, JBC Papers in Press, June 24, 2010, DOI 10.1074/jbc.M110.105080

Tomohiro Itoh‡1, Shu Takeda§, and Yukihiro Akao‡¶

From the ‡Gifu International Institute of Biotechnology, 1-1 Naka-Fudogaoka, Kakamigahara, Gifu 504-0838, the §Division ofEndocrinology, Metabolism and Nephrology, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, and the ¶UnitedGraduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

MicroRNAs (miRs) represent a class of endogenous �18–25nucleotide RNAs that regulate gene expression through transla-tional repression by binding to a target mRNA. These miRs reg-ulate several biological functions, such as cell growth, cell differ-entiation, carcinogenesis, and so on. In a previous report, wehave indicated that miR-141 and -200a act as preosteoblast dif-ferentiation modulators. In the present study, using microRNAarray and in silico analyses, we found that miR-208 is closelyinvolved in preosteoblast differentiation by partially regulatingthe expression of Ets1 (V-ets erythroblastosis virus E26 onco-gene homolog 1), which transactivates osteopontin, runt-re-lated transcription factor 2, parathyroid hormone-related pro-tein, and type I procollagen. Furthermore, the enforced expressionofmaturemiR-208 inmurine preosteoblast inMC3T3-E1 cells orprimary osteoblast cells remarkably attenuatedBMP-2-inducedpreosteoblast differentiation. In addition, we determined thatEts1 is a target gene of miR-208 by using a sensor luciferasereporter assay. Taken together, these results suggest that thedown-regulation of miR-208 in BMP-2-stimulated osteoblastdifferentiation is an important part of the regulatorymachineryinvolved in early osteogenesis.

MicroRNAs (miRs)2 represent a class of short (18–25 nucle-otide) noncoding single-stranded RNA molecules and haveemerged as a new class of regulators in both animal and plantdevelopment (1–3). Mature miRs regulate the repression ofspecific target gene translation and/or promote the degradationof their transcribed mRNAs by binding to the 3�-untranslatedregions (UTR) of the target genes. Recently,miRswere found tohave implications in diverse biological processes, including cellgrowth, cell differentiation, apoptosis, carcinogenesis, and dia-betes (4–8). In a previous study, we indicated that both miR-141 and -200a regulate BMP-2-stimulated preosteoblastMC3T3-E1 cell differentiation by directly targeting Dlx5 (dis-tal-less homeobox 5) (9). To date, it has already been reported

that several miRs, such asmiR-26a, miR-29,miR-133,miR-135,miR-206, andmiR-2861regulateosteoblastdifferentiation (10–14). Thus, it has been strongly suggested that the regulation ofosteoblast differentiation bymiRs is a notable component of theregulatory machinery.In this study, we aimed to uncover other miRs critical to

osteoblast differentiation. After we had investigated the miRexpression profiles in BMP-2-treated MC3T3-E1 cells bymiR array analysis, the expression level of miR-208 in BMP-2-treated MC3T3-E1 cells was significantly decreased comparedwith non-BMP-2-treated cells. To determine the role of miR-208 in osteogenesis, we performed a bioinformatics study andWestern blot analysis. In silico analyses showed that Ets1 (V-etserythroblastosis virus E26 oncogene homolog 1) could be a tar-get gene of miR-208.The purpose of the present study is to examine the regulation

of Ets1 expression by miR-208 in BMP-2-stimulated preosteo-blast differentiation in MC3T3-E1 cells. In the present study,we determined that Ets1 is a target gene of miR-208 by using asensor luciferase reporter assay. Our findings strongly suggestthat miRs regulate osteogenic master transcriptional factors inBMP-2-stimulated osteoblast differentiation.

EXPERIMENTAL PROCEDURES

Materials—Bioactive recombinant human BMP-2 was ob-tained from BioVision Research Products (Mountain View,CA). Eagle’s �-minimal essential medium, OPTI-MEM, FBS,and LipofectamimeTMRNAiMAXwere obtained from Invitro-gen. Alkaline phosphatase (ALP) assay kit LabAssayTM ALPwas fromWAKOPureChemical Industries Ltd. (Osaka, Japan).An ALP staining kit (TRAP and ALP double-stain kit) wasobtained from Takara Bio Inc. (Ohtsu, Japan). The antibodiesto mouse Ets1, runt-related transcription factor 2 (Runx2),osteopontin (OPN), parathyroid hormone-related protein(PTHrP), and ALP were obtained from Santa Cruz Biotechnol-ogy (Santa Cruz, CA).Cell Culture—Themurine preosteoblast cell lineMC3T3-E1

was obtained from RIKEN Cell Bank (Tsukuba, Ibaraki, Japan).Primary murine osteoblast cells, isolated from ICR mouse cal-varia, were purchased from Primary Cell Co. Ltd. (Sapporo,Hokkaido, Japan). These cells were cultured in phenol red-free�-minimal essential medium supplemented with 10% heat-in-activated FBS, 100 units/ml of penicillin, and 100 �g/ml strep-tomycin in a humidified atmosphere of 5%CO2 at 37 °C. BMP-2

* This work was supported by Grant-in-Aid for Young Scientists (B) 21700715from the Ministry of Education, Culture, Sports, Science and Technology.

1 To whom correspondence should be addressed. Tel.: 81-583-71-4646; Fax:81-583-71-4412; E-mail: [email protected].

2 The abbreviations used are: miR, microRNA; ALP, alkaline phosphatase;BMP, bone morphogenetic protein; OPN, osteopontin; PTHrP, parathyroidhormone-related protein; RNF11, ring finger protein 11; Runx2, runt-re-lated transcription factor 2; UTR, untranslated region(s); qRT, quantitativeRT.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 36, pp. 27745–27752, September 3, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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dissolved in PBS was added to the culture medium at a concen-tration of 300 ng/ml.MicroRNA Array Hybridization—Total RNA was extracted

from the cells by TRIzol containing phenol/guanidium isothio-cyanate (Invitrogen) with DNase I treatment.We examined theexpression profiles of miRs using Genopal�-MICH chips (Mit-subishi Rayon Co. Ltd., Yokohama, Japan), which is equippedwith 180 oligonucleotide DNA probes in hollow plastic fibersfor detection of mouse miRs. Hybridization signals were ana-lyzed by using a DNA chip analyzer according to the manufac-turer’s instructions.Quantitative RT-PCR—To confirm the reproducibility of

miR expression profiles, examined by miR array analysis,we measured their expression levels by using a TaqMan�microRNA reverse transcription kit and a TaqMan� microRNAassay kit (Applied Biosystems, Foster City, CA). Briefly, afterthe RT of 12.5 ng of total RNA, cDNAwas synthesized. The RTproducts were subjected to qRT-PCR using a Thermal CyclerDice� real time PCR system TP800 (Takara Bio). The expres-sion levels were normalized to U6, which was used as an inter-nal control and measured by the comparative Ct (��Ct)method. The qRT-PCR consisted of 45 cycles (95 °C for 10 s,60 °C for 40 s, and 72 °C for 1 s) after an initial denaturation step(95 °C for 10 min).To determine the level of Ets1 mRNA, we prepared cDNA

from total RNA samples using a PrimeScriptTM reagent kit(Takara Bio). qRT-PCR was performed (Thermal Cycler Dice�TP800) using a SYBER� Premix TaqTM II kit (Takara) and thefollowing primer sets: Ets1 sense, 5�-CTCTCCAGACAGAC-ACCTTGC-3�; Ets1 antisense, 5�-AGCACGGTCACGCAC-ATA-3�; Runx2 sense, 5�-GACGTGCCCAGGCGTATTTC-3�; and Runx2 antisense, 5�-AAGGTGGCTGGGTAGTGCA-TTC-3�. The cDNA of glyceraldehyde-3-phosphate dehydro-genase (GAPDH) was used as an internal control. The semi-quantitative RT-PCR consisted of 20 cycles (94 °C for 30 s,57.5 °C for 30 s, and 72 °C for 30 s) after an initial denaturationstep (94 °C for 4 min). The PCR products were analyzed byelectrophoresis on 2% agarose gels.Transfection of MC3T3-E1 Cells with miRNAs—MC3T3-E1

cells were seeded in 6-well plates at a concentration of 1 � 105cells/ml/well on the day before transfection. Themature type ofmiR-208 (Applied Biosystems; AUAAGACGAGCAAAAAGC-UUGU; see Fig. 1A) and its antisense inhibitor (Applied Biosys-tems)—designed to bind to endogenous specificmiRNAswhenintroduced into cells and inhibit their activities—were trans-fected using cationic liposomes (RNAiMAX) according tothe manufacturer’s lipofection protocol. The transfectionefficiency was evaluated by the transfection of the cells usingAlexa Flour 488 (Molecular Probes); transfection efficiencywas greater than 80% (data not shown). Nonspecific controlmiR (Applied Biosystems) was used as a control for nonspecificeffects. The culture medium containing 10% FBS, 100 units/mlpenicillin, and 100 �g/ml streptomycin was changed at 12 hafter the transfection. Transfected cells were cultured for 3 daysand then reseeded for various experiments at a concentration of1 � 105 cells/ml.ALP Activity and ALP Staining—MC3T3-E1 cells and the

transfectedMC3T3-E1 cellswere inoculated into 24-well plates

(1 � 105 cells/ml, 500 �l/well; Nunc, Roskilde, Denmark) andcultured with or without BMP-2, for 3 days. After incubation,the treated cells were washed twice with PBS, and 200�l of lysisbuffer was added to the cell layer and kept on ice for 5min. Thecell lysate was sonicated for 1 min and centrifuged at 1,000 � g,at 4 °C for 10 min. ALP activity was assayed by a spectrophoto-metricmethodusing a LabAssayTMALPkit (WakoPureChem-ical Industries Ltd., Osaka, Japan). The absorbance of each wellat 405 nm was measured with a microplate reader (Immuno-Mini NJ-2300; Nalge Nunc International K.K., Tokyo, Japan).Then ALP activity was estimated by the using the TRAP andALP double-staining kit (Takara) according to themanufactur-er’s protocol.Alizarin Red Staining—For detection of calcification in dif-

ferentiated MC3T3-E1 cells, the BMP-2-treated and non-treated cells were washed twice with PBS and fixed with 500 �lof ice-cold 70% ethanol for 10 min. The fixed cells were stainedwith 500 �l of Alizarin red solution (Sigma).Western Blotting—For preparation of cell lysate, MC3T3-E1

cells were washed twice with PBS and then harvested. The cellpellet was resuspended in radioimmune precipitation assaybuffer containing 25� Complete� and phosphatase inhibitormixture� (Roche Applied Science). Protein content was mea-sured with a DC protein assay kit (Bio-Rad). Each whole celllysate was resuspended in SDS-PAGE buffer containing 2%2-mercaptoethanol and was boiled at 98 °C for 5 min. Twentymicrograms of the protein from each cell lysate were separatedby SDS-PAGEby using a 12%polyacrylamide gel andwere elec-troblotted onto a PVDF membrane (GE Healthcare). Afterblockage of nonspecfic binding sites for 1 h by 5%nonfatmilk inTPBS (PBS and 0.1% Tween 20), the membrane was incubatedovernight at 4 °C with various primary antibodies. The mem-brane was then washed three times with TPBS, incubated fur-ther with horseradish peroxidase-conjugated secondary anti-body at room temperature, and washed again three times withTPBS. Protein bands were detected with enhanced ECL kit (GEHealthcare) and chemiluminescence detector (LAS-4000;Fujifilm).LuciferaseAssay—Todetermine the target region ofmiR-208

in Ets1, we constructed three kinds of pGL3-Ets1/miR-208 sen-sor plasmids by inserting a candidate binding site in the 3�-UTRof Ets1 mRNA into the XbaI site of the pGL3 control vector(Promega, Madison, WI). The PCR primer sequences used forconstruction of the pGL3-Ets1 sensor plasmids are as follows:pGL3-Ets1/miR-208 sensor-A sense, 5�-GCTCTAGAGC-CTTGAGTTGTGGATCTACTA-3�; pGL3-Ets1/miR-208sensor-A antisense, 5�-GCTCTAGAGCCCTTCAATCAAAG-TCCATCA-3�; pGL3-Ets1/miR-208 sensor-B sense, 5�-GCT-CTAGAGCTTTGATTGAAGGGGTCCA-3�; pGL3-Ets1/miR-208 sensor-B antisense, 5�-GCTCTAGAGCACCCCCT-GTCCCCAGATCAGCA-3�; pGL3-Ets1/miR-208 sensor-Csense, 5�-GCTCTAGAGCTACTGAGATCAGAGACCCAA-3�; and pGL3-Ets1/miR-208 sensor-C antisense, 5�-GCTC-TAGAGCGATTCCATAGAAGAGCTGCA-3�.Moreover, we constructed mutant vectors with deleted or

mutated miR-208 complementary sites in Ets1 by using aPrimeStarTM mutagenesis basal kit (Takara). The PCR primersequences used for construction of the mutated pGL3-Ets1/

MicroRNA-208 in BMP-2-induced Osteoblast Differentiation

27746 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 36 • SEPTEMBER 3, 2010


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miR-208 sensor-B plasmid are as follows: pGL3-Ets1/miR-208sensor-BDel sense, 5�-AAAAAATATATGAGGCCGCTCTA-AGGTGGTCT-3�; pGL3-Ets1/miR-208 sensor-B Del anti-sense, 5�-CCTTAGAGCGGCCTCATATATTTTTTTAA-GAG-3�; pGL3-Ets1/miR-208 sensor-B Mut sense, 5�-AATA-TCGTCGGCCGCTCTAAGGTGGTCT-3�; and pGL3-Ets1/miR-208 sensor-B Mut antisense, 5�-CCTTAGAGCGGCCG-ACGATATTTTTTTA-3�. MC3T3-E1 cells were inoculatedinto 12-well plates (1 � 105 cells/ml), co-transfected with thesensor vector plasmid or the mutated sensor vector, pRL-SV40vector, and 40 nM of miR-208 by using LipofectamimeTMRNAiMAX and were then cultured for 2 days. The luciferaseactivity was measured by using a luciferase assay system (Pro-mega) according to the manufacturer’s protocol. The relativeluciferase activity was expressed as the ratio of the measuredluciferase activity to the control (nonspecific miR).Statistical Analysis—All of the data were analyzed by one-

way analysis of variance and subsequently by Fisher’s multiplerange test among many groups and by Student’s t test betweentwo groups at the significance level of p � 0.05. Statistical anal-yses were carried out using StatView 5.0 software (SAS Insti-tute, Cary, NC).

RESULTS

Expression Profile of miR-208 in BMP-2-induced Preosteo-blast MC3T3-E1 Cell Differentiation—To investigate the ex-pression profiles ofmiRs inBMP-2-treatedMC3T3-E1 cells, weextracted each total RNA from BMP-2-treated and nontreatedcells at 24 h after treatment and subjected them to the Genopal�-MICH chips. The expression levels of miR-208 were signifi-cantly down-regulated (log ratio, �1.5) between BMP-2-treated and nontreated cells. To confirm the results of miRNAarray analysis, we examined the time-dependent expressionlevels of miR-208 in BMP-2-treated and nontreated cells byTaqMan� assay using a real timePCR. Interestingly, the expres-sion levels of miR-208 in BMP-2-stimulated cells were down-regulated as opposed to those in nontreated cells (Fig. 1B).Effects of miR-208 on Differentiation in MC3T3-E1 Cells—It

is well known that ALP activity increases in a time-dependentmanner inMC3T3-E1 cells after treatment with BMP-2 (1). Toinvestigate the role of miR-208 during differentiation, we mea-sured ALP activity after the transfection with miR-208 or anti-sense inhibitor for miR-208. As shown in Fig. 2, ALP activityand mineralization determined by Alizarin red staining in cellstransfected with miR-208 were suppressed. On the contrary,compared with BMP-2-treated MC3T3-E1 cells, ALP activityand mineralization in the antisense inhibitor remained un-changed (Fig. 2C, panel ii).Expression of Osteogenic Master Transcriptional Factor Ets1

Is Regulated byMicroRNA-208—To identify the target genes ofmiR-208 in osteogenesis, we searched for candidate genes usingthemiRBase Target version 5 andmiRanda databases. miR-208targets 823 genes, and osteogenic master transcriptional factorEts1 is included as a target gene (Fig. 3A). BMP-2 stimulationcaused a considerable increase in the protein expression levelsof Ets1 up to 48 h (Fig. 3B), whereas the levels of Ets1 mRNAexamined by real time PCR remained nearly unchanged from24–48 h (Fig. 3C). Furthermore, the levels of Runx2 and ALP

proteins that arewell known as osteoblast differentiationmark-ers were significantly increased by BMP-2 stimulation (Fig. 3B).However, the mRNA level of PTHrP, which is also modulatedby Ets1, was not changed by BMP-2 stimulation.The transfection of the cells with miR-208 induced the

down-regulation of Ets1 protein expression at the indicatedtimes (Fig. 4A, left panels), whereas the antisense inhibitor formiR-208 did not affect Ets1 protein expression (Fig. 4A, rightpanels). As judged by using a qRT-PCR, the levels of Ets1mRNA in the cells transfected with miR-208 remained un-changed compared with BMP-2-stimulated cells (Fig. 4B, panel(i)). Thus, the increase in Ets1 expression may be due to thedrop in the levels of miR-208 after the BMP-2 treatment (Fig.1B). These findings suggest that miR-208 regulates the expres-sion of Ets1 at a translational level.The protein expression levels of OPN, PTHrP, and Runx2

following BMP-2 stimulationwere further examined. As shownin Fig. 5, the expression level of OPN at day 10 was significantlydecreased in cells transfected with miR-208 compared withboth control miR-NC and BMP-2-stimulated cells. Addition-ally, the expression level of Runx2 at day 3 was slightlydecreased in cells transfected with miR-208 compared withboth control miR-NC and BMP-2-stimulated cells. On theother hand, the expression level of PTHrP, for which mRNAlevels are also regulated by Ets1, remained unchanged in thecells transfected with miR-208 (Fig. 5).Ets1 Is a Target of miR-208—To determine the target region

ofmiR-208 for Ets1mRNA,we constructed three kinds of lucif-erase reporter plasmids (A, B, and C) with or without the pos-sible binding region (numbers inMus musculus E26 avian leu-kemia oncogene 1, 5� domain (Ets1), transcript variant a,

FIGURE 1. Down-regulation of miR-208 in BMP-2-induced preosteoblastdifferentiation. A, the sequence of stem-loop structured miR-208. B, thechange of miR-208 expression levels in MC3T3-E1 cells until 18 days afterBMP-2 stimulation. MC3T3-E1 cells were incubated under BMP-2 stimulationat indicated times after the treatment, and then total RNA was extracted fromthe cells by TRIzol with DNase I treatment. Expression levels of miR-208 inBMP-2-treated cells were examined by TaqMan� microRNA assay using realtime PCR. The relative ratio is shown. The value of the cells before BMP-2treatment was designated as 1.




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FIGURE 2. Effects of transfection with mature and antisense inhibitor for miR-208 on differentiation in MC3T3-E1 cells. A, differentiation and mineral-ization in MC3T3-E1 cells transfected with miR-208 or antisense inhibitor for miRNA-208 were observed by ALP and Alizarin red staining after treatment withBMP-2. Negative control miR-NC (miR-NC) was designed to have no significant sequence similarity to mouse, rat, or human transcription products. B, theincreased expression of miR-208 in the cells by the transfection significantly suppressed the ALP activity. The cells were treated with BMP-2 (300 ng/ml) for 3days. The values of each group were expressed as the means � S.E. of three separate experiments. Mean values with different letters are significantly different(p � 0.01, one-way analysis of variance followed by Fisher’s multiple-range test). C, effects of miR-208 or antisense inhibitor for miRNA-208 on BMP-2-stimulated ALP activities in MC3T3-E1 cells. The cells transfected with miR-208 significantly suppressed the ALP activity following BMP-2 stimulation (panel i),but antisense inhibitor for miRNA-208 was unchanged compared with those of BMP-2-treated MC3T3-E1 cells (panel ii). The values of each group wereexpressed as the means � S.E. of three separate experiments.




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mRNA: 2246–2278) in 3�-UTR of Ets1 mRNA (Fig. 6A). Thelevels of luciferase activity in pGL3-Ets1/miR-208 sensor-B,which contains the 2246–2278 region of the 3-UTR of Ets1,were lower than those in the nonspecific control sensor vector(Fig. 6B). However, the levels of luciferase activity in pGL3-Ets1/miR-208 sensor-A/C, which lacks the 2246–2278 regionof the 3�-UTR of Ets1, were not decreased compared with thosein the nonspecific control sensor vector (p � 0.01). Weattempted to confirm these results by using two differentmutants of the miR-208-binding site in pGL3-Ets1/miR-208sensor-B (Fig. 6C). The levels of luciferase activity in cells trans-fected with the pGL3-Ets1/miR-208 sensor-B (wild type) weresignificantly reduced, whereas the activity level of sensor-Bmutants were not reduced (Fig. 6D). From these results, it ispossible that the target region of Ets1 (which we proposed) is abinding site for miR-208.Effects of miR-208 on Differentiation in Primary Murine

Osteoblast Cells—We examined the role of miR-208 during theBMP-2-induced osteoblast differentiation in primary murineosteoblast cells. As shown in Fig. 7, the ALP activities in cellstransfected with miR-208 were significantly suppressed. Theseresults suggest that the regulation of miR-208 in BMP-2-stim-ulated osteoblast differentiation is physiologically relevant.Effects of miR-208 on the Activation of ERK1/2—To gain

further insight into the mechanisms underlying regulation

FIGURE 3. Expression of Ets1 in BMP-2-stimulated osteoblast differentia-tion. A, nucleotide sequences of miR-208 and their complementation to Ets1.B, time course of Ets1, PTHrP, Runx2, and ALP expressions in BMP-2-treatedMC3T3-E1 cells (20 �g of protein/lane). A representative blot from three inde-pendent experiments is shown. C, time course of Ets1 and Runx2 mRNAexpressions in BMP-2-treated MC3T3-E1 cells. The values of each group wereexpressed as the means � S.E. of three separate experiments. Mean valueswith different letters are significantly different (p � 0.01, one-way analysis ofvariance followed by Fisher’s multiple-range test).

FIGURE 4. Expression of Ets1 is translationally regulated by miR-208 inosteoblast differentiation. A, expression profile of Ets1 protein in MC3T3-E1cells transfected with miR-208 or antisense inhibitor miR-208 after treatment(treatment time was 24, 48, and 72 h, 20 �g of protein/lane). A representativeWestern blot from three independent experiments is shown. B, panel (i), thechanges of Ets1 mRNA expression in the transfected cells with miR-208 wereexamined by qRT-PCR. GAPDH was used as an internal control. The values ofeach group were expressed as the means � S.E. of three separate experi-ments. Mean values with different letters are significantly different (p � 0.01,one-way analysis of variance followed by Fisher’s multiple-range test). Panel(ii), the relative level of Ets1 mRNA in the transfected cells was normalized toGAPDH. Lane 1, BMP-2 (�); lane 2, BMP-2(); lane 3, BMP-2() (300 ng/ml) andmiR-negative control; lane 4, BMP-2() and miR-208 (40 nM).

FIGURE 5. Expression of OPN, Runx2, and PTHrP in the cells transfectedwith miR-208 osteoblast differentiation. OPN, Runx2, and PTHrP expres-sion in BMP-2-treated MC3T3-E1 cells (20 �g of protein/lane). A representa-tive Western blot from three independent experiments is shown.




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of Ets1 expression by miR-208,we examined the activation ofERK1/2 in MC3T3-E1 cells andmiR-208-transfected MC3T3-E1cells. It is well known that ERK1/2regulates Ets1 activation (15). Thelevels of ERK1/2 activation byBMP-2 stimulation in miR-208-transfected MC3T3-E1 cells at theindicated times were almost similarto those ofMC3T3-E1 cells (Fig. 8,Aand B). Additionally, the levels ofERK1/2 activation by osteoblast dif-ferentiation medium stimulation inMC3T3-E1 cells also remainedunchanged compared with those ofMC3T3-E1 or miR-208-transfectedMC3T3-E1 cells (Fig. 8C).

DISCUSSION

In a previous study, we indicatedthat both miR-141 and -200a regu-late BMP-2-stimulated preosteo-blast MC3T3-E1 cell differentiationby directly targetingDlx5 (9). In thisstudy, we demonstrated for the firsttime that miR-208 regulates BMP-2-stimulated mouse preosteoblastdifferentiation by targeting Ets1. TheEts1 proto-oncogene, a member ofthe Ets family, was originally foundin a study of the avian E26 retrovirusgenome (15–17). The Ets transcrip-tion factor family is closely involvedin regulation of cell proliferation(18), differentiation (19), metastasis

(20), apoptosis (21), and angiogenesis (22). The Ets transcrip-tional factors also play critical roles in osteogenesis (23). Vary etal. (24) indicated that Ets1 is highly expressed during the pro-liferation stages in BMP-2-stimulated MC3T3-E1 cells. Ets1expression is alsomodulated by PKC, p38MAPK, retinoic acid,Ets1/AP-1 interaction, and the activation of the Ras/Raf/MEK/ERK1/2 signaling pathway. Consequently, this leads to thetransactivation of target genes, such as OPN, PTHrP, Runx2,and tenascin-C (a polymorphic high molecular mass extracel-lular matrix glycoprotein secreted from osteoblasts in osteo-genesis), and type I procollagen (15, 20, 25–31). The OPN andRunx2 expressions in miR-208-transfected MC3T3-E1 cellsfollowing BMP-2 stimulation were suppressed compared withthat of control cells, whereas the expression of PTHrPremained unchanged. As shown in Fig. 3B, PTHrP was con-stantly expressed in MC3T3-E1 cells and was not changed byBMP-2 stimulation. Thus, we consider that the expressionalregulations of OPN and Runx2 by miR-208 were preferentiallycarried out over PTHrP expression. This is likely because theexcess PTHrP expression is not a necessary response in BMP-2-stimulatedMC3T3-E1 cells. Therefore, the level of PTHrP inmiR-208-transfectedMC3T3-E1 cells might not have changed,

FIGURE 6. Identification of the target gene of miRNA-208 by luciferase reporter assay. A, schematic rep-resentation of the sensor vectors used in the luciferase assay for identification of target region in Ets1 formiR-208. Construction of three kinds (squares A, B, and C) of luciferase reporter plasmids including the 3�-UTRof Ets1 mRNA. B, each sensor vector, pRL-SV 40, and miR-NC or -208 were co-transfected into MC3T3-E1 cells.After 24 h, firefly and Renilla luciferase activities were measured by the using the luciferase assay system(Promega) according to the manufacturer’s protocol. The activities were calculated as a ratio of firefly to Renillaluciferase activity. The values of each group were expressed as the means � S.E. of three separate experiments.*, significant difference at p � 0.01 by Student’s t test. p � 0.05 is significant. C, the deleted or mutated regioncorresponding to the seed sequence of miR-208 sensor vector were made from pGL3-miR-208 sensor-B.D, MC3T3-E1 cells were co-transfected with the pGL3-Ets1/miR-208 sensor-B, deleted pGL3-Ets1/miR-208 sen-sor-B (Del), or mutated pGL-Ets1/miR-208 sensor-B (Mut) and miR-NC or -208. The activities were calculated as aratio of firefly to Renilla luciferase activity. The values of each group were expressed as the means � S.E. of threeseparate experiments. Mean values with different letters are significantly different (p � 0.01, one-way analysis ofvariance followed by Fisher’s multiple-range test).

FIGURE 7. Effects of transfection with mature and antisense inhibitorfor miR-208 on differentiation in primary murine osteoblast cells.A, the differentiation in primary murine osteoblast cells transfected with miR-208was observed by ALP staining after treatment with BMP-2. Negative controlmiR-NC (miR-NC) was designed to have no significant sequence similarity tomouse, rat, or human transcription products. B, the increased expression of miR-208 by the transfection significantly suppressed the ALP activity. The cells weretreated with BMP-2 (300 ng/ml) for 3 days. The values of each group wereexpressed as the means � S.E. of three separate experiments. Mean values withdifferent letters are significantly different (p � 0.01, one-way analysis of variancefollowed by Fisher’s multiple-range test).




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either. The enforced expression of miR-208 in MC3T3-E1 orprimary murine osteoblast cells significantly attenuated BMP-2-stimulated osteoblast differentiation, suggesting that thedown-regulation of miR-208 is an important common phe-nomenon for osteoblast differentiation.In an attempt to elucidate the regulatory machinery under-

lying the modulation of BMP-2-stimulated preosteoblast dif-ferentiation by miR-208, we investigated its effect on ERK acti-vation in BMP-2-stimulated MC3T3-E1 cells. Ets1 containstwo phosphorylation sites: threonine 38 and an array of serineswithin the exon VII domain. Phosphorylation of threonine 38by ERK1/2 activates Ets1, whereas phosphorylation of the exonVII domain by calcium/calmodulin-dependent protein kinaseandmyosin light chain kinase inhibits Ets1 DNA binding activ-ity (7). In a recent report, Foulds et al. (30) also indicated thatRas/mitogen-activated protein kinase (MAPK) signaling acti-vates Ets1 and Ets2 by cAMP-responsive element-binding pro-tein-binding protein/p300 recruitment. The data presented inFig. 8 show that ERK1/2 was activated by BMP-2 stimulation in

either MC3T3-E1 cells or the miR-208-transfected cells. Additionally,the behavior of the activation ofERK1/2 in normal MC3T3-E1 cellswas similar to that of the miR-208-transfected cells. Li et al. (12)reported that miR-208 expression isdown-regulated by osteogenesis-in-ducing medium stimulation inMC3T3 cells. We also observed theactivation of ERK1/2 in MC3T3-E1cells, as well as BMP-2 stimulation(Fig. 8C) by this particular osteo-genesis-inducing medium stimula-tion. Thus, the activation of ERK1/2in the osteoblast differentiationstage was irrelevant to the intracel-lular levels of miR-208. From theseobservations, it is highly probablethat the down-regulation of OPNand Runx2 expression, which aretranscriptionally regulated in partby Ets1, were mainly due to thedirect suppression of Ets1 expres-sion by miR-208 at the translationallevel.Ets1 also regulates ring finger

protein 11 (RNF11) expression inosteogenesis in MC3T3-E1 cells(33–35). RNF11 enhances the Smad4-dependent TGF� signaling pathway(26). Moreover, Azmi et al. (36)reported that RNF11 interacts withthe Hect family of E3 ubiquitinligases, and Smad-ubiquitin regula-tory factors (Smurf) 1 and 2, whichare negative regulators of bothBMP and TGF� signaling path-ways. It is well known that Smurf1

regulates osteoblast differentiation by enhancing proteasomaldegradation of R-Smads and Runx2 (37). On the other hand,Smurf2 preferentially regulates Smad1 and 2 (32). Therefore, itwas presumed that Ets1/RNF11/Smurf signaling acts as one ofthe key mechanisms of osteogenesis (Fig. 9). As a consequence,it was considered that the down-regulation of miR-208 was acritical process in BMP signaling during osteoblast differentia-tion. However, further study is required to clarify the relation-ship between miRs and the BMP signaling pathway.In conclusion, we have found that miR-208 regulates BMP-2-

stimulated mouse preosteoblast differentiation by targeting Ets1.Thus,miR-208 shouldbeconsideredan important candidate as anosteoblast differentiationmolecular target for the development ofpreventive or therapeutic agents against osteogenic disorders.REFERENCES1. Ambros, V. (2003) Cell 113, 673–6762. Ambros, V. (2004) Nature 431, 350–3553. Shukla, L. I., Chinnusamy, V., and Sunkar, R. (2008) Biochim. Biophys.

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FIGURE 8. Changes in the activation of ERK1/2 by miR-208 during BMP-2 stimulation in MC3T3-E1 cells byWestern blot analysis. A, the levels of activated ERK by BMP-2 stimulation in MC3T3-E1 cells (5 �g protein/lane). B, the levels of activated ERK1/2 by BMP-2 stimulation in MC3T3-E1 cells transfected with miR-208. C, thelevels of activated ERK by osteoblast differentiation medium (phenol red-free �-minimal essential medium-including 100 �g/ml of l-ascorbic acid, 10 mM of �-glycerophosphate, and 10 mM of HEPES buffer) stimulationin MC3T3-E1 cells. A representative blot from three independent experiments is shown.

FIGURE 9. A scheme showing the regulation of miR-208 on BMP-2-stimulated osteoblast differentiation.The enforced expression of miR-208 significantly blocked BMP-2-stimulated preosteoblast differentiation by sup-pression of Ets1 expression at the translational level. Gao et al. (34) reported that RNF11 expression is regulated byEts1. RNF11 regulates Smurf-dependent R-Smad degradation and TGF� signaling by Smad4 activation.




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Tomohiro Itoh, Shu Takeda and Yukihiro Akao Erythroblastosis Virus E26 Oncogene Homolog 1etsby Directly Targeting V-

MicroRNA-208 Modulates BMP-2-stimulated Mouse Preosteoblast Differentiation

doi: 10.1074/jbc.M110.105080 originally published online June 24, 20102010, 285:27745-27752.J. Biol. Chem.

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