Archives of Oral Biology (2005) 50, 227—236

www.intl.elsevierhealth.com/journals/arob

Altered gene expression in human cleidocranialdysplasia dental pulp cells

Shuo Chen, Lori Santos, Yimin Wu, Rose Vuong, Isabel Gay,Jennifer Schulze, Hui-Hsiu Chuang, Mary MacDougall*

Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio,7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA

Accepted 7 October 2004

KEYWORDSCleidocranial dysplasia;Runx2;Microarray;Gene regulation;Real-time PCR;Human dental pulp

cells

* Corresponding author. Tel.: +1 210 567-3798;fax: +1 210 567 6603.

E-mail address: macdougall@uthscsa.edu (M. MacDougall).

0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights resedoi:10.1016/j.archoralbio.2004.10.014

Introduction

Cleidocranial dysplasia (CCD) is an autosomal domi-nant human disease affecting both bone and toothformation. The main symptoms of CCD patients are

Summary Cleidocranial dysplasia (CCD) is an autosomal dominant disorder char-acterised by defects of bone and tooth development. The dental manifestations inCCD patients include supernumerary teeth, delayed tooth eruption, tooth hypoplasiaand absence of cellular cementum formation. This disorder is associated with muta-tions in the osteoblast-specific transcription factor Runx2. To identify morphologicaland molecular alterations associated with CCD dental tissues, human primary dentalpulp cell cultures were established from age- and sex-matched CCD and normalpatients. Dental pulp cells were compared for general morphology, proliferationrates, and gene expression profiles using cDNA microarray technology. CCD pulp cellswere about four-fold larger than normal cells, however the normal pulp proliferationrates were two- and three-fold greater at time points tested than the CCD cells. Of the226 genes analysed by blot microarray, 18.6% displayed significant differences at leasttwo-fold in expression levels. This includes 25 genes (11.1%) that were up-regulated,while 17 (7.5%) that were down-regulated in the CCD cells as compared to the normalcells. Expression of selected genes was further verified by quantitative real-timepolymerase chain reaction (qRT-PCR). Comparison between the CDD and normal cellsrevealed that gene expression of cytokines and growth factors, such as leukemiainhibitory factor (LIF), interleukin-6 (IL-6) and transforming growth factor betareceptor II (TGF-bRII) and vascular endothelial growth factor B (VEGFB) were higherwhile bone morphogenetic protein 2 (BMP2) was lower in the CCD cells. Furthermore,potential Runx2 binding sites were found in all putative target gene promoters. Thisstudy suggests that in addition to bone and tooth cell differentiation, Runx2 may beinvolved in controlling cell growth during tooth development.# 2004 Elsevier Ltd. All rights reserved.

rved.

228 S. Chen et al.

open fontanelles, hypoplasia or aplasia of the cla-vicles, a wide public symphysis, and short stature.1—4 Additionally, dental disorders include supernumer-ary teeth, abnormal tooth eruption, tooth hypopla-sia and lack of cellular cementum formation. CCD isassociated with mutations of Runx2, a transcriptionfactor essential for osteoblast and dental cell dif-ferentiation as well as bone and tooth formation.5

Mice with targeted disruption of Runx2 gene�/�

show no osteoblast differentiation with a completelack of bone formation and exhibit changes in earlydental development.6,7 Tooth germs are arrested atthe cap/early bell stages with no odontoblast cyto-differentiation and dentin matrix formation.8

Recently, studies have demonstrated that Runx2 isalso involved in fetal and postnatal growth of bone9

and postnatal formation of dental tissues and tootheruption.10,11

Runx2 (also known as Cbfa1, Osf 2, Pebp2aA orAML-3) belongs to the runt-domain gene familysince its DNA binding domain shares a high degreeof homology with the Drosophila pair-rule gene,runt.12 Runx2 specifically recognises a core DNAsequence and heterodimerises with the Cbfb unit,a cofactor that enhances affinity of Runx2 toDNA.13,14 Runx2 controls transcription of manybone- and tooth-related genes through its bindingsite.5,15—21 In addition, Runx2 is regulated bybone morphogenetic proteins (BMPs)/transforminggrowth factor b (TGF-b) family22—25 and othergrowth factors,8,26 by the mitogen-activated pro-tein kinase pathways,27 and by Smad proteins thatare signal transductors.28—30 Besides being a keydeterminant of the osteoblast lineage and differ-entiation as well as bone formation, recent studieshave shown that Runx2 regulates genes related tocell growth and controls osteoblast proliferation.31—36 Dentin and bone share several characteristics atmolecular levels and contain mineralised collagenmatrices and non-collagenous proteins, such asosteocalcin (OC), bone sialoprotein (BSP), dentinsialophosphoprotein (DSPP) and alkaline phospha-tase (ALP). It is proposed that cell-matrix interac-tions shown to be crucial for osteoblastdifferentiation are also necessary for odontogen-esis. Both odontoblast and dental pulp cells origi-nate from the dental papilla mesenchyme derivedfrom cranial neural crest cells. Several in vitro andin vivo studies have demonstrated that dental pulpcells are capable of differentiating into odonto-blasts and producing a mineralising matrix, particu-larly during reparative dentinogenesis.37,38 In situhybridisation studies have shown differentialexpression of Runx2 isoforms (types I—II) in mousedeveloping teeth with expression of all forms inodontoblast and dental pulp cells.21

In this study, we analysed basic cell propertiesand broadly surveyed the gene expressionprofiles of human primary dental pulp cellsisolated from a CCD versus normal patient. Over200 human genes, cytokines and receptors involvedin various biochemical pathways, were analysed aspotential Runx2 downstream target genes using amicroarray assay. Genes identified as potentiallyregulated by Runx2 were confirmed by qRT-PCRanalysis and a bioinformatics approach throughidentification of putative Runx2 binding sites in theirpromoters.

Materials and methods

Cell lines

Human primary normal (Runx2+/+) and CCD(Runx2+/�) dental pulp cells were established fromextracted teeth of a CCD patient as well as a normalage- and sex- matched control with signed informedconsent as previously outlined.39 The patient withCCD was an 11-year-old male and showed clavicularhypoplasia, short stature, low nasal bridge, andsupernumerary teeth as described previously byour laboratory.40 The genotype of the CCD patientshowed that arginine at position 225 was replacedwith glutamine (R225Q) within the COOH terminalend of the Runt domain. Runx2+/+ and Runx2+/� pulpexplants were grown at 37 8C in alpha minimalessential medium (a-MEM), containing 50 mg/mlascorbic acid, 10 mM Na b-glycerophosphate sup-plemented with 10% fetal bovine serum (FBS) and100 units/ml of penicillin/streptomycin andexpanded. Cells were observed and photographedusing a Nikon inverted microscope.

Cell growth analysis

Runx2+/+ and Runx2+/� cells were maintained in a-MEM supplemented with 50 mg/ml ascorbic acid,10 mM Na b-glycerophosphate, 10% FBS and100 units/ml of penicillin/streptomycin. Cells atthe 4th passage (p4) were seeded in six-well platesat 0.5 � 105 cells/plate. The growth media waschanged every 2 days and the cells cultured untilconfluency. Cell growth rates were assessed using aCoulter Counter by direct cell counting (BeckmanCoulter Inc. Fullerton, CA). For measurement ofthese cell volumes, the two cell populations(1 � 105 cells) at p4 were harvested from 135 cm2

wells, and spun down. The supernatant wasremoved carefully and cell pellets obtained. Then,100 ml of Dulbecco’s Phosphate-Buffered Saline(PBS) was added to the cell pellets to resuspend

Altered gene expression in human cleidocranial dysplasia dental pulp cells 229

the cells. The whole volume (cells plus 100 ml ofPBS) was measured from the two pulp cells. Totalcell volume from the normal and CCD cells wasobtained by the whole volume minus 100 ml ofPBS. Volume ratio of the CCD cells to the normalcells was determined by dividing the total CCD cellvolume by the total normal cell volume. This experi-ment was performed in triplicate wells.

RNA Isolation

Total RNA was isolated from the Runx2+/+ andRunx2+/� cells at p4 before cell confluency(70-80% cell density) using AtlasTM RNA isolationprotocol (NucleoSpin1 User Manual, Clontech,Palo Alto, CA). Briefly, cells were collected, lysedand then loaded into NucleoSpin1 columns andcentrifuged. The total RNA was eluted with nucle-ase-free water and treated with DNase I. Followingincubation at 37 8C for 15 min, 10� terminationmix (0.1 M EDTA, pH 8.0, 1 mg/ml glycogen)was added. Each sample underwent a phenol-chloroform extraction according to the protocol(Clontech). The top aqueous layer was removedafter centrifugation and transferred to a microcen-trifuge tube to which 1/10 volume of 2 M sodiumacetate, 2.5 volumes of 95% ethanol and 20 mg ofglycogen was added. The mixture was incubated onice for 10 min, centrifuged at 14,000 rpm at 4 8C for15 min, the supernatant discarded, and the pelletwashed with 75% ethanol. The samples were cen-trifuged again under the previous conditions and thesupernatant discarded. The pellet was air dried andresuspended in RNase-free water. The quality oftotal RNA was verified by running a RNA electro-

Table 1 Primers used for QRT-PCR.

Gene name Sequences

Cyclophilin forward 50 ggt gac ttcreverse 50 cat ggc ctc

TGF-bRII forward 50 ctg ctg cctreverse 50 gac atc ggt

BMP2 forward 50 cag acc accreverse 50 aag aag aat

TGF-b2 forward 50 gag tgc ctgreverse 50 gta gcg ctg

LIF forward 50 tgc caa tgcreverse 50 gcc aca tag

IL-6 forward 50 cct tcc aaareverse 50 tga ttt tca c

VEGFB forward 50 cga tgg cctreverse 50 gct cgg gta

phoresing agarose gel and integrity of the RNA wasconfirmed by the presence of 28S and 18S rRNAbands.

Microarray and image processing

Probe synthesis and purification were performedaccording to the protocol in the AtlasTM cDNAExpression Arrays User Manual (Clontech). Briefly,cDNA from the total RNA was reversely transcribedby MMLV reversed transcriptase in a reactionbuffer, containing [a-33P] dATP and the cDNA synth-esis (CDS) primer mixer which is a mix of primersspecific for a human cytokine/receptor array pro-vided by AtlasTM cDNA Expression Array kit (Clon-tech). The labelled cDNA was purified fromunincorporated 33P-labelled nucleotides by columnchromatography and hybridised to the human cyto-kine/receptor microarray (Human Cytokine/Recep-tor Array, PT3551-3). After hybridisation, the arrayswere exposed to a phosphrimaging screen (Molecu-lar Dynamics, Inc., Sunnyvale, CA). After scanning,the relative amounts of gene transcripts were deter-mined by measuring the isotope signal intensityratio.

Microarray data analysis

The arrays were aligned on a grid template todetermine the location of all genes on the arrays.Data normalisation, data filtering and pattern iden-tification were analysed by AtlasImageTM 2.0software (Clontech). The software was used tobalance the signals and to normalize possible dif-ferences in the RNA quantity. The signal intensities

References

aca cgc cat aa 30 43

cac aat att ca 30

gtg tga ctt tg 30 44

ctg ctt gaa gg 30

ggt tgg aga 30 45

ctc cgg gtt gtt t 30

aac aac gga tt 30 46

ggt tgg aga t 30

cct ctt tat tc 30 44

ctt gtc cag gt 30

gat ggc tga aa 30 47

ca ggc aag tct 30

gga gtg tgt 30 48

ccg gat cat 30

230 S. Chen et al.

Figure 1 Comparison of cell morphology and proliferation rates between Runx2+/� and Runx2+/+ dental pulp cells. (A)Phase contrast micrographs showing the cell morphology of human primary Runx2+/+ (A and B) and Runx2+/� (C and D)cells. (B) Cell proliferation rate between the two dental pulp cells. The Runx2+/+ and Runx2+/� cells (0.5 � 105 cells/plate) were grown in 135 mm2 wells. Cell numbers were counted at days 2, 4 and 8 of culture. The cell numbers wereplotted as a graph with the data showing mean � S.E. from three independent samples.

were normalised to the mean intensity of allthe genes represented on the array. At leasttwo-fold differences of gene expression profilesbetween the two groups were considered signifi-cant.

Quantitative real-time polymerase chainreaction (QRT-PCR)

The total RNA used for QRT-PCR was reversely tran-scribed using random hexamers and MultiScribereverse transcriptase according to manufacturer’sinstructions (ABI TaqMan Kit, Applied Biosystems,Foster City, CA). Amplification reactions were ana-

lysed in real-time on an ABI 7500 (Applied Biosys-tems) using SYBR Green chemistry and the thresholdvalues were calculated using SDS2 software (AppliedBiosystems). Thermal cycling parameters were95 8C for 30 s and 60 8C for 1 min, 40 cycles. Reac-tions were performed in quadruplicate and thresh-old cycle numbers were averaged. A single meltcurve peak was observed for each sample used indata analysis, confirming the purity and specificityof all amplified products. The threshold data gen-erated was normalised to cyclophilin A, which is apseudogene that has demonstrated minimal fluctua-tion in various tissue samples.41 Expression foldchanges (CCD/normal) were calculated according

Altered gene expression in human cleidocranial dysplasia dental pulp cells 231

to the formula2ðRt�EtÞ=2ðRn�EnÞwhereRt is the thresh-old cycle number for the housekeeping gene (cyclo-philin A) from the CCD cells and Et is the thresholdnumber for the experimental gene observed in theCCD cells. Rn is the threshold value for the house-keepinggene in thenormal cells (cyclophylinA)andEnis the threshold cycle number for the experimentalgene in the normal cells. The primer sequences usedfor real-time PCR are shown in Table 1.

Gene promoter analysis

Gene promoter analysis was performed for potentialRunx2 binding sites as described42 and following theexisting database programs: http://www.genoma-trix.de/cgi-bin/eldorado/main.pl; http://bimas.dcrt.nih.gov/molbio/signal.

Figure 2 Comparative gene expression intensity ofRunx2+/+ and Runx2+/� probes on a cDNA microarray. TotalRNAs from Runx2+/+ and Runx2+/� cells at the 4th passagewere extracted and converted to 33P-lableled cDNAprobes and hybridised with nylon membranes, containinghuman cytokine/receptor array following by the manu-facturer protocol (Clonetch). After hybridization, themembranes were washed and autoradiographied. Thehybridization intensity profiles were determined by phos-phorimager scanning. The images of results were analysedby AtlasImageTM 2.0 software (Clontech). A backgroundvalue around gene signals from each of the array wascalculated and subtracted from total gene signal. Arrowsshow the control housekeeping genes. The ratio of geneexpression in Runx2+/� and Runx2+/+ cells was calculated.

Results

Cell morphology and growth rates ofRunx2+/+ and Runx2+/� dental pulp cells

We first observed the basic cell morphology ofRunx2+/+ and Runx2+/� dental pulp cells using phasecontrast microscopy (Fig. 1A). Compared to thenormal pulp cells, Runx2+/� cells appear flat andlarger. Comparison of cell volume measurementsshowed that the volume of the Runx2+/� cells wasabout four-fold greater than the Runx2+/+ cells (datanot shown). In contrast, the cell proliferation rate ofthe normal pulp cells was two- and three-foldgreater compared to that of the Runx2+/� cells atthe various time points tested (Fig. 1B). These dataindicate that Runx2 is involved in regulating growthof human primary dental pulp cells.

Microarray analysis of gene expressionbetween Runx2+/+ and Runx2+/� cells

Among the 226 gene transcripts tested on the genearray (Fig. 2), expression levels ranged from a 59.5-fold under-expression to a 38.2-fold over-expressionin Runx2+/� cells. 25 genes (11.1%) were up-regu-lated at least two-fold while 17 (7.5%) showed atwo-fold or greater down-regulation in Runx2+/�

cells as compared to Runx2+/+ cells (Table 2). Wenoticed that gene expression showed significantdifferences within function groups between thetwo cell groups. More than 20 genes encoding trans-membrane proteins and their receptors were over-and under-expressed in these cells. For example,transforming growth factor-b receptor II (TGF-bRII)and vascular endothelial growth factor B (VEGFB)exhibited 32.7- and 4.5-fold increases, respectively,

whereas bone morphogenetic protein 2 (BMP2)showed a 2.2-fold decline in the Runx2+/� cells.Furthermore, different expression levels of cytokinegenes between the Runx2+/+ and Runx2+/� cellswere observed in the microarray.

Verification of microarray results by QRT-PCR

To further verify the microarray data, we selectedsix genes, including BMP2, LIF, IL-6, VEGFB, TGF-bRIIand TGF-b2 with altered expression levels and con-firmed their levels of expression using QRT-PCR. Theresults shown in Fig. 3 indicate that LIF, IL-6, VEGFBand TGF-bRII were 4.6-, 6.5-, 3.4- and 34.1-foldhigher, respectively, while BMP2 was 1.5-fold lowerin Runx2+/� cells. TGF-b2 was unaffected (data notshown).

232 S. Chen et al.

Table 2 Microarray analysis of gene expression in Runx2+/� cells compared to Runx2+/+ cells.

Fold " up-regulated gene Fold # down-regulated gene

38.2 Interleukin-6 (IL-6) �2.1 Brain-derived neurotrophic factor34.1 Transforming growth factor

beta receptor II (TGF-bRII)�2.1 BIGH3

29.3 Leukemia inhibitory factor (LIF) �2.2 Bone morphogenetic protein (BMP) 226.0 Metallothionein III �2.2 Wingless-related MMTV integration site 5a protein23.4 Interleukin-8 �2.3 Interferon-gamma (IFN-l) antagonist20.9 Glial cell-derived neurotrophic factor �2.3 Endothelin receptor type B12.0 Secreted apoposis related protein 1 �2.5 IFN-l receptor7.3 Macrophage inflammatory protein 2-a �2.7 Stem cell factor precursor7.3 Vascular endothelial growth factor C (VDGFC) �2.8 IGFBP complex acid labile chain5.2 Ribonuclease/angiogenin inhibitor �3.2 Fibroblast growth factor 75.0 Vascular endothelial growth factor (VDGF) �3.3 Insulin-like growth factor-binding protein24.8 Frizzled homology 2 �3.6 Interleukin 1 receptor type 14.5 Vascular endothelial growth factor B (VDGFB) �4.4 Transforming growth factor (TGF)-b23.9 Glia-derived neurite-promoting factor �4.7 Stromal cell-derived factor 13.5 Bone-derived growth factor 1 �5.1 Pleiotrophin (osf1)3.1 Ephrin type-B receptor 2 �38.3 Insulin-like growth factor binding protein 52.8 Acidic fibroblast growth factor �59.5 Neurotrophic tyrosine kinase receptor-related 32.7 INF-gamma receptor beta subunit2.7 Hepatocyte growth factor agonist/antagonist2.6 Inhibin beta A subunit2.4 Prohibitin2.3 Connective tissue growth factor2.2 Glial growth factor2.2 Placenta growth factor 12.1 Macrophage migration inhibitory factor

Identification of potential Runx2 bindingsites in promoters of target genes

To identify whether the promoters of these fivepotential downstream genes contained putativeRunx2 binding sites, a bioinformatics approachwas taken examining DNA sequence databases and

Figure 3 Comparison of differentially expressed genes betchanges for QRT-PCR were determined by dividing the individuexpression in the normal cells. The calculation method is de

programs (42 and http://www.genomatrix.de/cgi-bin/eldorado/main.pl; http://bimas.dcrt.nih.gov/molbio/signal). The results show that at least onepotential Runx2 site was found within the promoterof all five genes (Table 3). This data suggests thatthese genes are downstream target genes of Runx2and are regulated by Runx2 in pulp cells.

ween Runx2+/� and Runx2+/+ Cells by QRT-PCR. The foldal gene expression in the CCD cells by the individual genescribed in Section 2.

Altered gene expression in human cleidocranial dysplasia dental pulp cells 233

Table 3 Potential Runx2 binding sites.

Gene Sequence Reference

TGF-bRIIpromoter

as 50 TGTGGGT 30 49

50 AGTGGTG 30

50 TGCGGGG 30

s 50 AGTGGTG 30

50 AGTGGTG 30

VEGFBpromoter

as 50 GGTGGTT 30 50

s 50 TGCGGCT 30

50 TGTGGAG 30

IL-6 promoter as 50 AGCGGGT 30 51

s 50 AGTGGTG 30

BMP2 promoter s 50 TGCGGGG 30 52

LIF promoter as 50 AGCGGGT 30 53

50 TGTGGTG 30

s 50 TGCGGGG 30

Consensus sequence s 50 WGYGGKK 30 54,55

W, A or T; Y, C or T; K, G or T; as, antisense strand; s, sensestrand.

Discussion

In the present study, we found that the osteoblast-specific transcription factor Runx2 contributes tothe control of cell growth in human primary dentalpulp cells. The phenotype and genotype of thepatients with CCD used in this study have beendescribed earlier40 and shared some common char-acteristics as reported by other groups.1—4 Thesephenotypes include clavicular hypoplasia, short sta-ture, low nasal bridge, and supernumerary teeth. Amissense mutation at 225 (R225Q) within the Runtdomain in the CCD patient has also been found bytwo independent groups.3,4 This mutated Runx2protein was unable to quantitatively accumulatein the nucleus3 and failed to regulate its target geneactivity,4 suggesting that CCD in these patients iscaused by haploinsufficiency.

In this study, we found that Runx2+/� cells have analtered morphology and decreased proliferationrate as compared to Runx2+/+ cells. Based on theseobservations, we used a human cytokine/receptorarray to determine the pattern of gene expressionbetween the Runx2+/� and Runx2+/+ cells usingmicroarray technology. This technique provides apowerful tool to analyse the expression levels oflarge numbers of genes in a single experiment. Ourdata analysis demonstrated that a sets of genesinvolved in alterations between the Runx2+/� andRunx2+/+ cells include cell signalling and growth. Weselected five genes for verification: LIF, IL-6, VEGFB,TGF-bRII and BMP2. LIF and IL-6 belong to pleiotro-

pic cytokines expressed in multiple tissues and havediverse biological functions, such as bone cell meta-bolism and embryo development.56 Palmqvistet al.57 reported that IL-6 and IL-6 receptor increaseexpression of NF-kB ligand (RANKL) and osteopro-tegerin (OPG), but decrease RANK expression inneonatal mouse calvaria. The same regulation wasobserved in themouse calavaria by LIF. In addition toosteoclastogenesis, IL-6 and LIF also promote com-mitted osteoblast differentiation and bone forma-tion. IL-6 in combination with its receptor inducesmurine embryonic fibroblast differentiation towardthe osteoblast lineage and stimulates expression ofALP and OC.58 Additionally, LIF enhances basic fibro-blast growth factor and increases IL-6 synthesis viaJanus family tyrosine kinase2-signal transducer andactivator of transcription 3 (JAK2/STAT3) pathwaysin osteoblast-like MC3T3-E1 cells.59 In this study, wefound higher expression levels of IL-6 and LIF genesin Runx2+/� cells using microarray and QRT-PCR, andidentified Runx2 sites in these two gene promoterregions. However, mechanisms by which the hetero-zygous mutation of Runx2 resulted in increasedexpression of IL-6 and LIF as well as a decrease inpulp cell proliferation are unknown. Furthermore,the relationship between IL-6, LIF and cell prolif-eration is also unclear. Although Forst et al.60

describe that IL-13 increases the levels of IL-6 geneexpression in human osteoblast-like cells but inhi-bits their cell proliferation rates, it remains obscurewhether IL-6 directly affects dental pulp cell growthin relationship to the heterozygous mutation ofRunx2.

We also observed that several growth factorswere up- and down-regulated in Runx2+/� cells. Thisincludes TGF-bRII, VEGFB and BMP2. TGF-b is apotent multiple functional regulator of cell growthand differentiation. In particular, members of theTGF-b superfamily with important roles on bone andtooth cell differentiation are the BMPs. TGF-b andBMPs bind to distinct receptors, TGF-b receptors Iand II for TGF-b and BMP receptors I and II for BMPs.Following ligand binding, receptor-associatedkinase is activated and phosphorylates Smads, whichtranslocate into the nucleus to interact with Runx2and regulate the transcription of target genes.28—30

Ji et al.34 reported that Runx2 regulates the TGF-breceptor I gene promoter. We provided an additionalexample in that Runx2 is able to up-regulate BMP2and to down-regulate TGF-bRII and VDGFB in humanprimary dental pulp cells. Also, potential Runx2sites exist in these gene promoters. Although theregulation of Runx2 on these growth factor genesare not known, both negative and positive regula-tion of target genes by Runx2 is likely dependent onthe context of Runx2 binding sequence within the

234 S. Chen et al.

promoter, the cell-type aswell as the interactionwithother co-factors.23,28—30 For example, Javed et al.16

describe the suppression of BSP by Runx2 as cell type-independent. They propose that the context of Runx2sequences within the promoter region contributes tothe formation of a Runx2 regulatory complex, func-tioning as either a repressor or activator. Allistonet al.24 found that Runx factors inhibit transcriptionof the OC gene in rat osteosarcoma (ROS 17/2.8) andmouse embryonic fibroblast (C3H-10T1/2) cells, butenhance its activity inhumanhepatic tumour (HepG2)cells mediated by Smad3 and TGF-b. They suggestthateffects ofRunx factors on target genesdependonthe cell type and promoter. In addition, studies havedemonstrated that the coordinated action of Runx2and other factors are required for osteogenic differ-entiation and other events.29 Runx2 cooperates withthe c-myc gene in promoting T-cell growth and for-mation of T-cell lymphoma.31,32 However, calvarialcells from Runx2�/� mutant mice exhibit increasedcell growth rates.36 These data suggest that regula-tion of its downstream genes and control of cellgrowth by Runx2 as an inhibitor or enhancer maydepend on the physiological microenvironments thatdictate the biological functions of Runx2 in dental andnon-dental cells.

In summary, the present study demonstrates thata heterozygous Runx2 mutation changes cell mor-phology and decreases cell growth of human primarydental pulp cells. Gene expression involved in cellgrowth and signalling was significantly differentbetween the Runx2+/+ and Runx2+/� cells as deter-mined by DNA microarray and QRT-PCR. Differentgene expression patterns may reflect intrinsic func-tional differences of cell growth rates between thetwo cells. However, the molecular mechanisms needto be further investigated in the future.

Acknowledgments

This work was supported by National Institute Dentaland Craniofacial Research Grant DE 113221.

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