Krüppel-Like Zinc Finger Protein Glis3 Promotes OsteoblastDifferentiation by Regulating FGF18 Expression

Ju Youn Beak, Hong Soon Kang, Yong-Sik Kim, and Anton M Jetten

ABSTRACT: The zinc finger protein Glis3 is highly expressed in human osteoblasts and acts synergisticallywith BMP2 and Shh in enhancing osteoblast differentiation in multipotent C3H10T1/2 cells. This induction ofosteoblast differentiation is at least in part caused by the induction of FGF18 expression. This study supportsa regulatory role for Glis3 in osteoblast differentiation.

Introduction: Gli-similar 3 (Glis3) is closely related to members of the Gli subfamily of Krüppel-like zincfinger proteins, transcription factors that act downstream of sonic hedgehog (Shh). In this study, we analyzedthe expression of Glis3 in human osteoblasts and mesenchymal stem cells (MSCs). Moreover, we examinedthe regulatory role of Glis3 in the differentiation of multipotent C3H10T1/2 cells into osteoblasts and adipo-cytes.Materials and Methods: Microarray analysis was performed to identify genes regulated by Glis3 in multipotentC3H10T1/2 cells. Reporter and electrophoretic mobility shift assays were performed to analyze the regulationof fibroblast growth factor 18 (FGF18) by Glis3.Results: Glis3 promotes osteoblast differentiation in C3H10T1/2 cells as indicated by the induction of alkalinephosphatase activity and increased expression of osteopontin, osteocalcin, and Runx2. In contrast, Glis3expression inhibits adipocyte differentiation. Glis3 acts synergistically with BMP2 and Shh in inducing osteo-blast differentiation. Deletion analysis indicated that the carboxyl-terminal activation function of Glis3 isneeded for its stimulation of osteoblast differentiation. Glis3 is highly expressed in human osteoblasts andinduced in MSCs during differentiation along the osteoblast lineage. Microarray analysis identified FGF18 asone of the genes induced by Glis3 in C3H10T1/2 cells. Promoter analysis and electrophoretic mobility shiftassays indicated that a Glis3 binding site in the FGF18 promoter flanking region is important in its regulationby Glis3.Conclusions: Our study showed that Glis3 positively regulates differentiation of C3H10T1/2 cells into osteo-blasts and inhibits adipocyte differentiation. Glis3 acts synergistically with BMP2 and Shh in inducing osteo-blast differentiation. The promotion of osteoblast differentiation by Glis3 involves increased expression ofFGF18, a positive regulator of osteogenesis. This, in conjunction with the induction of Glis3 expression duringosteoblast differentiation in MSCs and its expression in osteoblasts, suggests that Glis3 is an importantmodulator of MSC differentiation.J Bone Miner Res 2007;22:1234–1244. Published online on May 7, 2007; doi: 10.1359/JBMR.070503

Key words: Gli-similar 3, mesenchymal stem cell, osteoblast, differentiation, BMP2, fibroblast growth factor18, adipocyte

INTRODUCTION

MESENCHYMAL STEM CELLS (MSCs) are nonhematopoi-etic stem cells present in bone marrow. These cells

are able to differentiate into multiple mesoderm cell lin-eages, including pre-osteoblasts, pre-chondrocytes, pre-adipocytes, and myoblasts, that play a central role in theformation of bone, cartilage, adipose tissue, and muscle,respectively.(1–4) Study of the regulation of proliferationand differentiation of MSCs into osteoblasts is important

for obtaining insights into the basic mechanisms that con-trol this process. In addition, such studies are relevant to thedevelopment of new therapeutic strategies for various dis-eases, including osteoporosis, and may promote the use ofstem cells in tissue engineering.(4,5)

The differentiation into each of these different lineages iscontrolled by specific transcription factors that control cellfate decisions.(1,6) Differentiation of MSCs along the osteo-blast lineage involves commitment into osteoblast progeni-tors and their maturation into osteoblasts. Several tran-scription factors have been reported to play a key role inthe regulation of this commitment, including runt-relatedThe authors state that they have no conflicts of interest.

Cell Biology Section, LRB, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutesof Health Research, Triangle Park, North Carolina, USA.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 22, Number 8, 2007Published online on May 7, 2007; doi: 10.1359/JBMR.070503© 2007 American Society for Bone and Mineral Research

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gene 2 (Runx2), also referred to as core binding factor � 1(Cbfa1), and the zinc finger protein osterix.(7–10) In addi-tion, a large number of signaling molecules have been im-plicated in the regulation or modulation of osteogenesis,including steroid hormones,(11) members of the TGF-�family, including several BMPs,(1,12–15) several fibroblastgrowth factors (FGFs),(16–18) and the Wnt and hedgehogsignaling pathways.(13,19,20)

Gli and Gli-similar (Glis) transcriptional factors form twoclosely related subfamilies of Krüppel-like zinc finger pro-teins.(21–23) These proteins share a highly conserved tandemrepeat of five C2H2-type zinc finger motifs that mediatetheir binding to Gli binding sites in the promoter of targetgenes. Gli proteins act downstream of sonic hedgehog(Shh) and exhibit critical functions in the regulation of em-bryonic development, including various aspects of skeletaldevelopment.(3,9,20,24–28) Shh has been reported to enhanceosteoblast differentiation in MSCs.(3,29–32)

Because Shh/Gli signaling has been implicated in skeletaldevelopment and MSC differentiation, we were interestedin examining the potential role of Glis3 in the differentia-tion of MSCs. Glis3 is an 84-kDa protein that contains re-pressor and activator domains, suggesting that it can func-tion both as an activator and repressor of transcription.(22)

Glis3 is expressed in many adult tissues. During mouse em-bryonic development. Glis3 is expressed in a temporal andspatial manner. Between E13.5 and E16.5 of mouse devel-opment, Glis3 is expressed in a dynamic pattern during limbdevelopment.(22) Recent studies have implicated Glis3 in arare syndrome with neonatal diabetes mellitus and hypo-thyroidism.(33)

In this study, we investigated the potential role of Glis3 inthe regulation of differentiation of multipotent C3H10T1/2cells into the osteoblast and adipocyte lineage. Under dif-ferent inducing conditions, these cells have been reportedto differentiate into adipocytes, myotubes, chondroblasts,and osteoblasts.(2,4,15,19,26,34,35) We show that Glis3 is highlyexpressed in human osteoblasts and induced during differ-entiation of MSCs into osteoblasts. Morover, Glis3 pro-motes differentiation of C3H10T1/2 cells into the osteoblastlineage and acts synergistically with BMP2. Our results in-dicate that Glis3 is a modulator of MSC differentiation andsuggest that the enhancement in osteoblast differentiationby Glis3 is at least in part mediated by increased expressionof FGF18.

MATERIALS AND METHODS

Cell culture

Multipotent C3H10T1/2 cells (ATCC) were grown inDMEM supplemented with 10% FBS and antibiotics(Gibco-BRL). Human MSC (hMSC) and mesenchymalstem cell growth factor medium (MSCGM) growth mediawere purchased from Cambrex, and human osteoblasts(hOB)(C-12720) and osteoblast growth medium were pur-chased from Promocell (Heidelberg, Germany). To induceosteoblast differentiation, cultures were maintained in me-dium supplemented with 1 mM L-glutamine, 10 mM �-glyc-erolphosphate, 50 �g/ml ascorbic acid, and in the case of

hMSCs, with 1 mM dexamethasone (osteogenic-promotingmedium). Cells were treated with or without BMP2 (Wy-eth), FGF18, or Shh (Sigma) as indicated. To neutralizeFGF18, cells were grown in medium containing anti-FGF18antibody (Santa Cruz Biotechnology).

Plasmids

The plasmid pM-Glis3 and the pTAL-LUC-(GlisRE)3

reporter plasmid were described previously.(22) pCMV-Flag-Glis3, pCMV-Flag-Glis3�N307, and pCMV-Flag-Glis3�C525 were generated by cloning, respectively, theentire coding sequence of Glis3, the region encoding thecarboxyl terminus from 307His to 774Gly, and the regionencoding the amino-terminus from 1Met to 525Pro into theEcoRI and BamHI sites of pCMV-3xFlag 7.1 (Sigma).These fragments were generated by PCR amplification us-ing pM-Glis3 DNA as template. The regions encoding3xFlag-Glis3, 3xFlag-Glis3�N307, and 3xFlag-Glis3�C525were reamplified and subcloned into the NotI and BamHIsites of the retroviral vector pLXIN (BD Biosciences Clon-tech). A 5�-promoter flanking region of the mouse FGF18gene, extending from nt −1639 to nt +18, was generated byPCR using mouse genomic DNA. The PCR product wascloned into the vector pCRII-TOPO (Invitrogen) and sub-sequently inserted into the KpnI and XhoI restriction sitesof the luciferase reporter plasmid pGL4.10 Basic (Promega),thereby generating pGL4-FGF18(-1613). The promoter de-letion constructs pGL4-FGF18(−1258), pGL4-FGF18(−958),pGL4-FGF18(−658), and pGL4-FGF18(−358) were gener-ated by PCR using pGL4-FGF18(−1639) DNA as a tem-plate. Mutant pGL4-FGF18(−1639) containing point muta-tions in the Glis3-RE1 was generated using a Quickchangesite-directed mutagenesis kit (Stratagene). The sequence ofall plasmids was verified by restriction enzyme analysis andDNA sequencing.

Retroviral infection

Retroviral infection and neomycin selection were carriedout as described previously.(36) After infection with retro-virus, C3H10T1/2 cells were selected in medium containingG418 (500 �g/ml). Resistant clones were pooled and arereferred to as C3H10T1/2-Empty, C3H10T1/2-Flag-Glis3,C3H10T1/2-Flag-Glis3�N307, and C3H10T1/2-Flag-Glis3�C525.

Subcellular localization

C3H10T1/2 cells were plated in glass bottom culturedishes (Mattek) and 24 h later were fixed and permeabi-lized as described.(37) Cells were subsequently incubatedfor 2 h with mouse anti-FLAG M2 antibody (Sigma), andfinally for 40 minutes with goat anti-mouse Alexa Fluor594antibody (Molecular Probes). Cells were washed with PBSand nuclei stained with 4�-6-diamidino-2-phenylindole(DAPI). Fluorescence was examined in a Zeiss confocalmicroscope LSM 510 NLO confocal microscope.

Alkaline phosphatase assays

To measure alkaline phosphatase (ALP) activity, cellswere washed three times with cold PBS and sonicated inlysis buffer A (10 mM Tris, 2 mM MgCl2, 0.05% Triton-

REGULATION OF OSTEOBLAST DIFFERENTIATION BY Glis3 1235

X100, pH 8.2). After centrifugation, supernatant was col-lected and assayed for ALP substrate using 4-nitrophenylphosphate as substrate. ALP staining was performed withan ALP staining kit from Sigma according to the manufac-turer’s instructions.

Adipocyte differentiation

C3H10T1/2 cells were grown to confluence and treatedwith insulin (1 �g/ml), dexamethasone (0.25 �g/ml), andisobutylmethylxanthine (IBMX; 0.5 mM). After 24-h incu-bation, medium was changed and cells were maintained inmedium containing 1 �g/ml insulin. At day 7, cells werestained with 0.3% Oil-Red-O(38) or collected for RNA iso-lation.(37) Northern blot analysis was carried out as de-scribed previously(37) using a [32P]labeled probe for fattyacid binding protein 4 (FABP4 or aP2).

Microarray analysis

Microarray analysis was carried out by the NIEHS Mi-croarray Group (NMG) as described previously.(37) Geneexpression analysis was conducted using Agilent mouseoligo arrays (Agilent Technologies) representing ∼20,000genes. Total RNA was isolated from C3H10T1/2-Emptyand C3H10T1/2-Flag-Glis3 using Qiagen RNeasy Mini Kit.The microarray data discussed in this study have been de-posited in the NCBI’s Gene Expression Omnibus (GEO,http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?)and are accessible through GEO Series accession numberGSE5379.

Real-time QRT-PCR analyses

Real-time QRT-PCR reactions were carried out in trip-licate in a 7300 Real Time PCR system using the TaqManOne-Step RT-PCR mix (Applied Biosystems) as describedpreviously(37) with predesigned Assays-on-Demandprimers/probe set for mFGF18 (Mm00433286_m1) andhGlis3 (Hs00541450_m1) (Applied Biosystems). Otherprimers and probes were designed using the Primer Express2.0 software. F: 5�-CGGCTACCACATCCAAGGAA, R:5 � -GCTGGAATTACCGCGGCT, and 5 � -FAM-TGCTACCAGACTTGCCCTC-TAMRA for 18S rRNA;F: 5�-GGCATTGCCTCCTCCCTC, R: 5�-GCAGGCTG-TAAAGCTTCTCC, 5�-FAM-CGGTGAAAGTGACT-GATTCTGGCAGCTC-TAM for mouse osteopontin;F : 5 � - G G C C T T C A A G G T T G T A G C C C , R : 5 � -CCCGGCCATGACGGTA, and 5�-FAM-CCACA-GTCCCATCTGGTACCTCTCCG-TAMRA mRunx2;F: 5�-GTTCAGGGTGTGT-CGTCGAAC, R: 5�-TTTCGGCTCGACGGTCTCAAA for mouse osteocalcin.F: 5 � -CTCTACAGCCGGACCAGTG-3 � , R: 5 � -CCGAAGGTGTCTGTCTCCAC-3� for hFGF18;F: 5�-CTCACTACCACACCTACCTG-3� , R: 5�-TCAATATGGTCGCCAAACAGATTC-3� for hRUNX2;F: 5�-TTGCAGTGATTTGCTTTTGC-3�, R: 5�-ACACT-ATCACCTCGGCCATC-3� for human osteopontin. All re-sults were normalized to the 18S transcript that served as aninternal control.

Reporter gene assays

Cells were plated in 12-well dishes at 5 × 104 cells/welland 20 h later co-transfected (0.5 �g total DNA in 1 ml)with 0.1 �g reporter plasmid, 0–0.3 �g of pCMV-Flag-Glis3plasmid, and 0.05 �g pCMV�−galactosidase, which servedas an internal control to monitor transfection efficiency.Cells were transfected in Opti-MEM (Life Technologies)and 1.5 �l Fugene 6 transfection reagent (Roche). Cellswere incubated for 30 h and assayed for �-galactosidase andluciferase activity. Luciferase activity was assayed with aluciferase kit (Promega). The level of �-galactosidase activ-ity was determined using a luminescent �-galactosidase de-tection kit (Clontech) according to the manufacturer’s in-structions. Transfections were performed in triplicate, andeach experiment was repeated at least twice.

EMSA

E. coli BL21(DE3) transformed with pQE32-Glis3(ZFD),encoding the DNA binding domain from 307His to 525Ser,was grown at 37°C to midlog phase and treated with iso-propyl-�-D-thiogalactopyranoside (0.5 mM final concentra-tion) for 3 h (His)6-Glis3(ZFD) was purified using Ni-NTA+ resin (Qiagen). (His)6-Glis3(ZFD) recombinantprotein (0.2 �g) was incubated in binding buffer (20 mMHEPES, pH 8.0, 5% glycerol, 2.5 mM MgCl2, 1 mM DTT,50 mM KCl, 10 mM ZnSO4, 300 mg poly(dI-dC) with[32P]labeled, double-stranded oligonucleotides for 40 minat room temperature as described previously.(22) The pro-tein–DNA complexes were separated on a 10% nativepolyacrylamide gel and visualized by autoradiography.

RESULTS

Characterization of C3H10T1/2-Flag-Glis3 cell lines

To study the potential regulatory role of Glis3 on thedifferentiation of multipotent C3H10T1/2 cells, we ex-pressed Flag-Glis3 and two Glis3 mutants Flag-Glis3�N307and Flag-Glis3�C525, containing an amino- or carboxyl-terminal deletion, respectively, in these cells and examinedtheir effects on osteoblast and adipocyte differentiation. Asshown in Fig. 1A, examination of the expression of thesefusion proteins by Western blot analysis showed that theanti-Flag antibody recognized a protein of the expected sizein all three cell lines. However, in lysates from cells express-ing Flag-Glis3 and Flag-Glis3�C525, the anti-Flag antibodydetected an additional protein of ∼36 kDa. This truncatedFlag-Glis3 protein seems to be an amino-terminal fragmentof Glis3 generated by proteolytic processing of full-lengthFlag-Glis3. Examination of their subcellular localizationshowed that all three proteins were largely confined to thenucleus (Fig. 1B). To analyze the transcriptional activity ofthe Glis3 proteins, C3H10T1/2 cells were transiently trans-fected with pTAL-(GlisRE)3-LUC plasmid DNA, in whichthe luciferase reporter is under the control of three Glis3binding sites (GlisRE). Full-length Glis3 and Glis3�N307induced transcription several-fold (Fig. 1C), indicating thatthey act as positive regulators of gene transcription and thatthe amino-terminus is not required for this function. Flag-

BEAK ET AL.1236

Glis3�C525 did not enhance transcription in agreementwith previous observations showing that Glis3 mutants witha carboxyl-terminal deletion act as dominant-negative tran-scription factors.(21)

Glis3 acts synergistically with BMP2 to induceosteoblast differentiation

To examine the effect of Glis3 on osteoblast differentia-tion, C3H10T1/2-Empty and C3H10T1/2-Flag-Glis3 cellswere grown to confluence and cultured further in differen-tiation medium in the absence or presence of BMP2. After4 days of incubation, cells were stained for ALP, a markerfor osteoblast differentiation.(12,30) As shown in Figs. 2Aand 2C, cultures of untreated C3H10T1/2-Empty cellsstained weakly for ALP, whereas cultures of C3H10T1/2-Flag-Glis3 cells exhibited an increased number of positivelystained cells. Treatment with 100 ng/ml BMP2 enhancedALP staining in both C3H10T1/2-Empty and C3H10T1/2-Flag-Glis3 (Fig. 2B) in agreement with previously reportedosteoblast-stimulating activity of BMP2.(3) The highestnumber of ALP+ cells was observed in cultures ofC3H10T1/2-Flag-Glis3 cells treated with BMP2 (Fig. 2D).This was confirmed by analysis of ALP activity (Figs. 2Eand 2F). BMP2 induced ALP activity in C3H10T1/2-Emptyand C3H10T1/2-Flag-Glis3 cells in a time- and concentra-tion-dependent manner. An increase in ALP activity wasobserved at 3 days after the addition of 100 ng/ml of BMP2,whereas a concentration of 50 ng/ml BMP2 was sufficient toinduce ALP activity. ALP activity was consistently inducedto higher levels in C3H10T1/2-Flag-Glis3 than C3H10T1/2-Empty cells in agreement with the conclusion that Glis3expression promotes the induction of osteoblast differen-

tiation by BMP2. This conclusion was supported by experi-ments examining the effect of Glis3 on the expression oftwo other osteoblast-specific markers, Runx2 (Cbfa1) andosteocalcin (OCN). Runx2 mRNA expression was inducedsignificantly by BMP2 but only slightly by Glis3 (Fig. 3A);little synergism was observed between BMP2 and Glis3. Incontrast, Glis3 and BMP2 had a synergistic effect on OCNmRNA expression (Fig. 3B) as shown for ALP.

Shh has also been reported to promote osteoblast differ-entiation, suggesting a role for Shh/Gli signaling in the regu-lation of MSC differentiation.(3,30–32) Figure 2G shows thatGlis3 expression enhanced the induction of ALP activity byShh. Shh had little effect on Glis3 expression (Fig. 2H) anddid not affect the transcriptional activity of Glis3 (notshown).

Effect of Glis3 mutants on osteoblast differentiation

We next examined the effect of two mutants, Glis3�N307and Glis3�C525, on osteoblast differentiation (Figs. 3C and3D). As expected, Glis3�N307, which as full-length Glis3acts as a transactivator, induced ALP and osteopontin(OPN) expression in C3H10T1/2 cells. In contrast,Glis3�C525, which does not contain the transactivation do-main and acts as a dominant-negative repressor, inhibitedrather than enhanced ALP activity. These data show thatthere is a correlation between the transactivating activity ofGlis3 and its mutants and their ability to promote osteoblastdifferentiation and indicates that the activation domain atthe carboxyl terminus of Glis3 is essential for its ability toenhance osteoblast differentiation.

FIG. 1. Subcellular localization and trans-activating activity of Glis3 and mutant Glis3proteins in C3H10T1/2-Flag-Glis3, C3H10T1/2-Flag-Glis3�C525, and C3H10T1/2-Flag-Glis3�N307 cells. (A) Proteins were isolatedfrom C3H10T1/2 cells and examined by West-ern blot analysis using an anti-Flag M2 anti-body. A schematic view of the three fusionproteins is shown above the Western blot.AD, activation domain; ZDF, zinc-finger do-main; FL, full-length. (B) Subcellular localiza-tion of Flag-Glis3 was examined by confocalmicroscopy using mouse anti-Flag M2 anti-body. Nuclei were identified by DAPI stain-ing. (C) Comparison of the transactivating ac-tivity of Glis3 and mutant Glis3 proteins.C3H10T1/2 cells were co-transfected with(GlisRE)3-LUC, pCMV�, and either pCMV-3xFlag-Glis3, pCMV-3xFlag-Glis3�N307, orpCMV-3xFlag-Glis3�C525 as indicated. Af-ter 30 h, cells were assayed for luciferase(LUC) and �-galactosidase activity. The rela-tive LUC reporter activity was calculated andplotted.

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Expression of Glis3 inhibitedadipocyte differentiation

Because Glis3 promoted osteoblast differentiation inmultipotent C3H10T1/2 cells, we were interested in deter-mining whether Glis3 expression had any effect on the dif-ferentiation of these cells along a different lineage, such asadipocyte differentiation. As shown in Fig. 4, treatmentC3H10T1/2-Empty cells with IBMX induced adipocyte dif-ferentiation as indicated by increased Oil red O stainingand FABP4/aP2 mRNA expression, markers of adipocytedifferentiation. In contrast, the induction of adipocytemarkers was suppressed in C3H10T1/2 cells expressing ei-ther Glis3 or Glis3�N307, suggesting that these cells have agreatly reduced ability to undergo adipocyte differentia-tion. In contrast, adipocyte differentiation was enhancedrather than inhibited in C3H10T1/2 cells expressingGlis3�C525, which functions as a dominant-negative regu-lator of transcription.(22) These results show that expressionof transcriptionally active forms of Glis3 favor osteoblastdifferentiation and inhibit adipocyte differentiation.

Identification of Glis3-regulated genes

To study changes in gene expression resulting from theexpression of Glis3, we performed microarray analysis us-ing RNA from late exponential phase cultures of C3H10T1/2-Flag-Glis3 and C3H10T1/2-Empty cells. This analysisidentified a number of genes that were up- or downregu-lated in C3H10T1/2-Flag-Glis3 cells compared with controlcells (Table 1). Genes upregulated by Glis3 included demi-lune cell and parotid protein (Dcpp), a gene encoding asecretory protein with little known function, the imprintedgene mesoderm-specific transcript (MEST),(39) the tran-

scription factors GATA-binding protein 2 (GATA2) andCCAAT/enhancer binding protein delta (C/EBP�), and thegrowth factors IGF-1 and FGF18. Several genes encodingmembrane and extracellular matrix proteins were repressedby Glis3, including procollagen type IX � 1 (COL9a1), te-nomodulin (TMD), fibromodulin (FMOD), extracellularmatrix protein 1 (ECM1), and disintegrin-like and metallo-proteinase 2 (ADAMTS2). Because FGF18 has been re-ported to promote osteogenesis,(17,40,41) we hypothesizedthat induction of osteoblast differentiation by Glis3 couldbe mediated by increased expression of FGF18. We firstconfirmed the induction of FGF18 by Glis3 by QRT-PCRanalysis. As shown in Fig. 5A, C3H10T1/2-Flag-Glis3 cellsexpressed 12-fold higher levels of FGF18 mRNA thanC3H10T1/2-Empty cells. Treatment of C3H10T1/2 andC3H10T1/2-Flag-Glis3 cells with BMP2 did not change theexpression of FGF18 mRNA or Glis3 significantly (data notshown). As shown in Fig. 5B, Glis3�N307 was also able toinduce the expression of FGF18 mRNA, whereasGlis3�C525 did not have any significantly effect. Thus, theinduction of FGF18 expression correlated with the tran-scriptional activity exhibited by the mutants.

Next, we examined whether FGF18 was able to promoteosteoblast differentiation in C3H10T1/2 cells. Figure 5Cshows that FGF18 treatment enhanced osteoblast differen-tiation in C3H10T1/2 cells in a concentration-dependentmanner as indicated by the increase in ALP activity. More-over, addition of FGF18 neutralizing antibody to the cul-ture medium greatly inhibited the induction of ALP activity(Fig. 5D). These observations further support a role ofFGF18 in the promotion of osteoblast differentiation byGlis3.

FIG. 2. Glis3 expression enhances osteoblast differentiation in C3H10T1/2 cells. C3H10T1/2-Empty (Control) and C3H10T1/2-Flag-Glis3 cells were grown to confluence and cultured in differentiation medium with and without BMP2 (100 ng/ml). Four days later, cellswere stained for alkaline phosphatase (ALP) (A–D). (E) Confluent C3H10T1/2-Empty (Con) and C3H10T1/2-Flag-Glis3 cells werecultured in differentiation medium with and without BMP2 (100 ng/ml). At the time intervals indicated, cells were collected and assayedfor ALP activity. (F) Confluent C3H10T1/2-Empty and C3H10T1/2-Flag-Glis3 cells were maintained in differentiation medium in thepresence of BMP2 at the concentration indicated. After 4 days of incubation, cells were assayed for ALP activity. (G) Confluentcultures of C3H10T1/2-Empty and C3H10T1/2-Flag-Glis3 cells were maintained in differentiation medium with or without Shh (1�g/ml). At the indicated time, cells were collected and assayed for ALP activity. (H) C3H10T1/2-Empty cells were treated with orwithout Shh and then assayed for Glis3 mRNA expression by real-time QRT-PCR.

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Next, we examined whether Glis3 expression correlatedwith the expression of FGF18 in human osteoblasts andduring osteoblast differentiation in MSCs. Figure 5E showsthat expression of Glis3, FGF18, RUNX2, and OPN wereincreased significantly during osteoblast differentiation inMSC and that all four genes were highly expressed in hu-man osteoblasts. The significantly greater expression ofthese genes in osteoblasts compared with differentiatedMSC might be caused by the inefficient osteogenic differ-entiation in MSCs. These data are in agreement with theconcept that Glis3 and FGF18 play a role in osteoblastdifferentiation

Induction of FGF18 promoter activity by Glis3

To examine whether the expression of FGF18 might beregulated by Glis3, we searched the −1639-bp 5�-promoterflanking region of the mFGF18 gene for the presence ofpotential Glis3 binding sites. Two potential Glis3 bindingsites were identified at −1438 (Glis3-RE1) and −746 (Glis3-RE2; Fig. 6A). We examined the ability of Glis3 to activatea luciferase reporter gene under control of the FGF18 pro-moter using a series of promoter deletion constructs (Fig.6B). Expression of Glis3 induced a 12-fold increase in tran-scriptional activation with the −1639 proximal promoter re-gion. This transactivation by Glis3 was significantly reducedwith the −1258 promoter deletion construct, in which Glis3-RE1 was deleted. Further deletion of Glis3-RE2 (−958,−658, and −358) caused only a small decrease in transcrip-tional activation. These results suggest that the Glis3-RE1might be important in the regulation of FGF18 expressionby Glis3.

To determine whether Glis3 was able to bind Glis3-RE1and Glis3-RE2, we examined the binding of Glis3(ZFD) to[32P]labeled Glis3-RE1, Glis3-RE2, and Gli-RE oligo-nucleotides by EMSA. As shown in Fig. 6C, Glis3 was ableto interact with Glis3-RE1 (Fig. 6C, lane 2) but failed tointeract with Glis3-RE1mut (lane 5) and Glis3-RE2 (lane10). Addition of unlabeled Glis3-RE1 oligonucleotidescompeted effectively for Glis3 binding (lane 3). Binding of

FIG. 3. Effect of Glis3 on the induction of ALP, Runx2, OCN,and OPN expression. C3H10T1/2-Empty (Con), C3H10T1/2-Flag-Glis3 (Glis3) cells were induced to differentiate with or with-out BMP2 for 3 or 6 days before the expression of Runx2 (A) andOCN mRNA (B) were analyzed by QRT-PCR. (C and D)C3H10T1/2-Empty, C3H10T1/2-Flag-Glis3, C3H10T1/2-Flag-Glis3�N307, and C3H10T1/2-Flag-Glis3�C525 cells were inducedto differentiate in the presence of 100 ng/ml BMP2. Four or 10days later, cells were assayed for, respectively, ALP activity (C) orrelative OPN mRNA expression (D).

FIG. 4. Glis3 expression inhibits adipocyte differentiation.C3H10T1/2 cells were induced to differentiate into adipocytes andafter 7 days, cells were stained with Oil red O (A–D) or RNA wascollected to determine the level of FABP4/aP2 mRNA expressionby Northern blot analysis (E). (A) C3H10T1/2-Empty; (B)C3H10T1/2-Flag-Glis3 (FL); (C) C3H10T1/2-Flag-Glis3�N307;and (D) C3H10T1/2-Flag-Glis3�C525 cells. (Bottom panel) Levelof 18–28S rRNA.

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Fig 4 live 4/C

Glis3 to Gli-RE (lane 8 and 13) was used as a positivecontrol.

The role of Glis3-RE1 in the activation of theFGF18(−1639) promoter by Glis3 was further confirmed byanalysis of a FGF18(−1639) promoter mutant containingthe Glis3-RE1mut mutation. This mutation greatly inhib-ited the activation of FGF18(−1639) promoter by Glis3(Fig. 6D). These results are in agreement with the hypoth-esis that the induction of FGF18 mRNA expression by

Glis3 is regulated at a transcriptional level and involves aninteraction of Glis3 with Glis3-RE1.

DISCUSSION

In this study, we examined the potential regulatory roleof Glis3 and two Glis3 mutants on the differentiation ofmultipotent C3H10T1/2 cells into osteoblasts and adipo-cytes. We showed that full-length Glis3 and the mutant

TABLE 1. CHANGES IN GENE EXPRESSION INDUCED BY GLIS3 IN C3H10T1/2 CELLS

Gene name GenBank accession no. Description Fold increase

Dcpp NM_019910 Demilune cell and parotid protein 52.9Mest NM_008590 Mesoderm specific transcript 50.7ApoD NM_007470 Apolipoprotein D 5.8SCRN1 AK129084 Secernin 1 5.2Ramp2 NM_019444 Receptor activity modifying protein 2 5.1Igf1 NM_010512 Insulin-like growth factor 1 4.5GATA2 NM_008090 GATA-binding protein 2 4.4Ahr NM_013464 Aryl-hydrocarbon receptor 4.2Nkd1 NM_027280 Naked cuticle 1 homolog 4.1Ltbp2 NM_013589 Latent TGF-� binding protein 2 4Lcn2 NM_008491 Lipocalin 2 3.8Dspg3 NM_007884 Dermatan sulphate proteoglycan 3 3.7Pcp4 NM_008791 Purkinje cell protein 4 3.7Lum NM_008524 Lumican 3.6Hp NM_017370 Haptoglobin 3.5Gpm6b NM_023122 Glycoprotein m6b 3.3Vil2 NM_009510 Villin 2 3.2GTL2 Y13832 Transient receptor cation channel 3.1Gas6 NM_019521 Growth arrest specific 6 2.9Cyp2f2 NM_007817 Cytochrome P450 2.8Prss11 NM_019564 Protease, serine, 11 2.5Ctgf NM_010217 Connective tissue growth factor 2.4Cebpd NM_007679 C/EBPdelta 2.2Wnt5b NM_009525 Wingless-rel. MMTV integration 5B 1.8Wisp2 NM_016873 WNT1 inducible protein 2 1.7Fgf18 NM_008005 Fibroblast growth factor 18 1.4Rrm1 NM_009103 Ribonucleotide reductase M1 −1.6Pcolce2 NM_029620 Procollagen C-endopeptidase enhancer 2 −1.6Chst7 NM_021715 Carbohydrate sulfotransferase 7 −1.6TRIP13 AK010336 Thyroid hormone receptor interactor 13 −1.6Cyp26b1 NM_175475 Cytochrome P450, 26b1 −1.6Fst NM_008046 Follistatin −1.6TMEM23 NM_144792 Transmembrane protein 23 −1.7Dhrs3 NM_011303 Dehydrogenase/reductase −1.7Adamts2 NM_175643 Disintegrin-like and metalloprotease −1.8Ecm1 NM_007899 Extracellular matrix protein 1 −1.8Tm4sf6 NM_019656 Transmembrane 4 superfam. member 6 −1.8Sdpr NM_138741 Serum deprivation response −1.9Hsd11b1 NM_008288 Hydroxysteroid 11-� dehydrogenase 1 −2.1Xlr NM_011725 X-linked lymphocyte-regulated complex −2.1Tgfbi NM_009369 TGF-� induced −2.3Fmod NM_021355 Fibromodulin −2.6EPHA7 NM_010141 Eph receptor A7 −2.6Olfr437 NM_146296 Olfactory receptor 437 −3Scx NM_198885 Scleraxis −3TNMD NM_022322 tenomodulin −3.1Col9a1 NM_007740 Procollagen, type IX, � 1 −3.3KRTAP3–1 D86424 Keratin associated protein 3–1 −3.4Pem NM_008818 Placenta and embryo oncofetal gene −4.6

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Glis3�N307, in which the amino terminus up to the firstzinc finger is deleted, both localized to the nucleus andfunctioned as transcriptional activators in C3H10T1/2 cells.Glis3�C525, in which the activation domain at the car-boxyl-terminus is deleted, was unable to activate transcrip-tion. Previous studies showed that deletion of the activationdomain generates a Glis3 mutant that acts as a dominant-negative transcription factor.(21) Our data showed that thetranscriptional activation domain at the carboxyl terminusis critical for the transactivating activity of Glis3 inC3H10T1/2 cells.

More importantly, we showed that expression of full-length Glis3 promoted osteoblast differentiation inC3H10T1/2 cells as indicated by the induction of ALP ac-tivity, and increased expression of OCN and OPN mRNA.BMP2, and Shh, which by themselves induce osteoblast dif-ferentiation in MSCs(3,15,27,29,30,42) increased osteoblast dif-ferentiation much more efficiently in C3H10T1/2 cells ex-pressing Glis3 than in cells expressing empty vector. Ourobservations suggest that Glis3 expression promotes osteo-blast differentiation and acts synergistically with BMP2 andShh. Because BMP2 induces RUNX2 expression and Glis3does not, BMP2 may function as an initiator at an earlystage, whereas Glis3 may act at a later stage as a promoterof osteoblast differentiation. The Glis3�N307 deletion mu-tant was also able to enhance osteoblast differentiation.These results suggest that its amino-terminus is not neededfor this action of Glis3. The dominant-negative Glis3�C525mutant did not significantly increase osteoblast differentia-tion. These results indicate that the enhancement of osteo-blast differentiation requires the transactivation function ofGlis3.

To determine whether Glis3 affected the differentiationof C3H10T1/2 cells into other mesoderm cell lineages, weexamined the effect of Glis3 on adipocyte differentiation.Our data showed that Glis3 expression inhibited adipocytedifferentiation as indicated by reduced fat accumulationand expression of the adipocyte marker FABP4/aP2.Again, Glis3�N307 exhibited the same effect as full-lengthGlis3, whereas Glis3�C525 increased rather than decreasedadipocyte differentiation. These results show that Glis3 is anegative regulator of adipocyte differentiation and suggestthat Glis3 may have a role in controlling the differentiationof MSCs into different lineages or at least the differentia-tion into osteoblasts and adipocytes. Our observations arein agreement with previous studies showing a reciprocalrelationship between the differentiation of MSCs into adi-pocytes and osteoblasts.(3,43) Activation of the deacetylaseSirt1 in C3H10T1/2 cells also results in decreased adipocyteand increased osteoblast differentiation.(43) Similarly treat-ment with Shh enhances osteoblast and inhibits adipocytedifferentiation in MSCs.(3) Therefore, the induction of os-teoblast differentiation by Glis3 could be linked to Shh sig-naling. However, Glis3 was found not to affect Shh expres-sion nor does Shh alter Glis3 expression (Fig. 2H) or itsactivation (data not shown), suggesting a different mecha-nism of action. The reciprocal relationship between differ-entiation of MSC in adipocyte and osteoblast differen-tiation is relevant to human disease. In age-related osteo-porosis, cell populations in bone marrow are replaced

FIG. 5. Glis3 enhances the expression of FGF18 mRNA expres-sion in C3H10T1/2 cells. (A) RNA was isolated from exponentialphase (Exp) and confluent (Confl) cultures of C3H10T1/2-Empty(Con) and C3H10T1/2-Flag-Glis3 cells in the absence of BMP2and examined for FGF18 mRNA expression by real-time QRT-PCR analysis. (B) Comparison of FGF18 mRNA expression be-tween C3H10T1/2-Empty, C3H10T1/2-Flag-Glis3, C3H10T1/2-Flag-Glis3�N307, and C3H10T1/2-Flag-Glis3�C525 cells. Allcells were cultured for 5 days in differentiation medium (−BMP2)before cells were collected and assayed for ALP activity. (C) In-duction of osteoblast differentiation in C3H10T1/2 cells byFGF18. Confluent cultures were treated with FGF18 (10 or 50ng/ml) in differentiation medium for 5 days (−BMP2) before cellswere collected and assayed for ALP activity. (D) Effect of FGF18neutralizing antibody. C3H10T1/2-Empty (Con), C3H10T1/2-Flag-Glis3, C3H10T1/2-Flag-Glis3�N307 and C3H10T1/2-Flag-Glis3�C525 cells were cultured in the presence of anti-FGF18antibody at the indicated concentrations, and 4 days later wereassayed for ALP activity. (E) Elevated expression of RUNX2,Glis3, and FGF18 RNA in human osteoblasts and in hMSC in-duced to differentiate along the osteoblast lineage. Exponentialphase cultures of hMSC and confluent cultures of hMSC (hMSC-D) or osteoblasts maintained for 2 wk in osteogenic promotingmedium were assayed for Glis3, RUNX2, FGF18, and OPN ex-pression by real-time QRT-PCR.

REGULATION OF OSTEOBLAST DIFFERENTIATION BY Glis3 1241

by adipose cells, suggesting a shift in the differentiation ofstem cells from the osteoblast to the adipose lineage.(44)

To obtain insight into the mechanism by which Glis3induces osteoblast differentiation, we assessed changes ingene expression in C3H10T1/2 cells induced by Glis3 usingmicroarray analysis. This analysis showed that Glis3 expres-sion repressed a number of membrane and extracellularmatrix genes, including FMOD, ECM1, COL9a1, TMD,and ADAMTS2. These results suggest a role for Glis3 in theregulation of extracellular matrix homeostasis. A numberof genes were found to be positively regulated by Glis3,including MEST, transcription factors C/EBP� andGATA2, and growth factors IGF-2 and FGF18. FGF18 is amember of the FGF family and mediates its action by bind-ing to FGF receptor 3 (FGFR3). FGF18 was of particularinterest because of its reported positive regulatory role inosteogenesis and chondrogenesis.(17,40,41,45) Bone develop-ment has been reported to be delayed in FGF18-deficientmice likely because of a decrease in the proliferation ofosteogenic mesenchymal cells and a delay in terminal dif-ferentiation to osteoblasts. Mice lacking FGF18 display de-layed ossification and decreased expression of osteogenicmarkers. In agreement with these studies, we showed thatFGF18 enhanced osteoblast differentiation in C3H10T1/2cells. Moreover, addition of FGF18 neutralizing antibodiesgreatly reduced osteoblast differentiation in C3H10T1/2-Flag-Glis3 cells. These data support our hypothesis that theincrease in osteoblast differentiation is at least in part

caused by the induction of FGF18 expression by Glis3.However, we cannot rule out that other mechanisms areinvolved such as interactions of Glis3 with other transcrip-tion factors that act downstream of BMP2. We furthershowed that differentiation of MSC along the osteoblastlineage is associated with increased expression of Glis3 andFG18 and that Glis3 and FGF18 expression is greatly el-evated in osteoblasts. These data are in agreement with theconcept that Glis3 and FGF18 have a regulatory role inosteoblast differentiation.

Our results showed that the induction of FGF18 expres-sion is dependent on the transactivation function of Glis3.These observations suggested that Glis3 might regulate thetranscription of FGF18 expression directly. Previous studieshave shown that Glis3 regulates transcriptional activationby interacting with specific DNA binding sites, containingthe consensus sequence GACCACCCA, in the promoterregulatory region of target genes (JY Beak, unpublishedobservations, 2007).(22) Examination of the −1639 bp 5�

promoter flanking region of the FGF18 gene identified twopotential Glis3 DNA response elements (Glis3-RE1 and−2). Promoter analysis showed that Glis3 activates tran-scription of a reporter gene under the control of a 1.6-kbFGF18 promoter region and that deletion or mutation ofGlis3-RE1 greatly reduced this activation, whereas deletionof Glis-RE2 had little effect, suggesting that Glis3-RE1, butnot Glis3-RE2, is important in the transcriptional activation

FIG. 6. Role of Glis3 response-elements (Glis3-REs) in the regulation of FGF18 expression by Glis3. (A) Schematic representationof 5�-promoter regulatory region of the FGF18 gene. The two potential Glis3 binding sites Glis3-RE1 and Glis3-RE2 are indicated. (B)Activation of the FGF18(−1639) promoter by Glis3. C3H10T1/2 cells were co-transfected with a series of pGL4.10 basic reporterplasmids containing progressively shorter fragments of the FGF18 5�-flanking region (0.1 �g), Glis3 expression plasmid (0.1 �g), andpCMV�−galactosidase (0.05 �g). Cells were assayed for luciferase and �-galactosidase activity, and the relative luciferase activity wasplotted. Results represent the mean ± SE of data from three independent experiments each performed in triplicate. (C) Binding of(His)6-Glis3(ZFD) to [32P]labeled Glis3-RE1, mutated Glis3-RE1 (Glis3-RE1mut), Glis3-RE2, and Gli-RE oligonucleotides wasassessed by EMSA in the presence or absence of unlabeled Glis3-REs (50× excess) as indicated. The sequence of each Glis3-RE isshown at the top right. (D) Effect of Glis3 on the activation of FGF18(−1639)mut-Luc carrying the Glis3-RE1mut mutation. C3H10T1/2cells were transfected with FGF18(−1639)-Luc, FGF18(−1639)mut-Luc, and Glis3 expression plasmid and processed as described under B.

BEAK ET AL.1242

of FGF18 by Glis3. The latter was supported by EMSAanalysis showing that Glis3 was able to bind Glis3-RE1 butnot Glis3-RE2.

In summary, our results show that the Krüppel-like zincfinger protein Glis3 promotes osteoblast and inhibits adi-pocyte differentiation and that these effects are dependenton the transactivation function of Glis3. In addition, weshow that Glis3 acts synergistically with BMP2 in inducingosteoblast differentiation. Glis3 induces changes in the ex-pression of several genes, including FGF18. We provideevidence indicating that Glis3 regulates FGF18 expressionby binding a specific Glis3 DNA binding site in its pro-moter. Our data indicate that Glis3 has a regulatory role inosteoblast differentiation and that its stimulation of differ-entiation is at least in part caused by the induction ofFGF18, a positive regulator of osteogenesis. Insights intothe signaling pathways that regulate Glis3 activity couldlead to new strategies in the treatment of osteoporosis.

ACKNOWLEDGMENTS

We thank Drs Jaemog Soh and John Roberts for com-ments on the manuscript. This research was supported bythe Intramural Research Program of the NIH, National In-stitute of Environmental Health Sciences.

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Address reprint requests to:Anton M. Jetten, PhD

Cell Biology SectionLRB Division of Intramural Research

National Institute of Environmental Health SciencesNational Institutes of Health

Research Triangle Park, NC 27709, USAE-mail: jetten@niehs.nih.gov

Received in original form August 29, 2006; revised form March 14,2007; accepted April 27, 2007.

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