216

ORIGINAL

BMP2-induced gene profiling in dental epithelial cell line
a

b

a

a

a

Keiko Miyoshi , Hideya Nagata , Taigo Horiguchi , Kaori Abe , Ivan Arie Wahyudi ,bca

Yoshinobu Baba , Hidemitsu Harada , and Takafumi Nomaa

Department of Molecular Biology, Institute of Health Biosciences, the University of Tokushima

Graduate School, Tokushima, Japan ; bDepartment of Applied Chemistry, Graduate School of Engineering, Nagoya University, Aichi, Japan ; and cDepartment of Oral Anatomy II, Iwate Medical
University School of Dentistry, Iwate, Japan
Abstract : Tooth development is regulated by epithelial-mesenchymal interactions andtheir reciprocal molecular signaling. Bone morphogenetic protein 2 (BMP2) is known as
one of the inducers for tooth development. To analyze the molecular mechanisms of BMP2
on ameloblast differentiation (amelogenesis), we performed microarray analyses using
rat dental epithelial cell line, HAT-7. After confirming that BMP2 could activate the canonical BMP-Smads signaling in HAT-7 cells, we analyzed the effects of BMP2 on 14,815
gene expressions and profiled them. Seventy-three genes were up-regulated and 28 genes
were down-regulated by BMP2 treatment for 24 hours in HAT-7 cells. Functional classification revealed that 18% of up-regulated genes were ECM/adhesion molecules present
in the enamel organ. Furthermore, we examined the expression of several differentiation markers in dental epithelial four cell-lineages including inner enamel epithelium(ameloblasts), stratum intermedium, stratum reticulum, and outer enamel epithelium.
The results indicated that BMP2 might induce at least two different cell-lineage markers including a BMP antagonist expressed in HAT-7 cells, suggesting that BMP2 could accelerate amelogenesis via BMP signaling. J. Med. Invest. 55 : 216-226, August, 2008
Keywords : dental epithelium, BMP2, microarray, gene profiling

INTRODUCTION
Tooth development is initiated in dental epithelial cells derived from ectodermal cells, and underlying ectomesenchymal cells that are subsequently
activated to promote the developmental cascade.
The reciprocal interactions between epithelial andmesenchymal cells are strictly regulated during
tooth development not only by growth factors andcytokines such as bone morphogenetic proteins(BMPs), fibroblast growth factors, Wnts, and tuReceived for publication April 10, 2008 ; accepted June 13,
2008.
Address correspondence and reprint requests to Takafumi Noma,
Department of Molecular Biology, Institute of Health Biosciences,
the University of Tokushima Graduate School, Kuramoto-cho,
Tokushima 770-8504, Japan and Fax : +81-88-633-7326.

mor necrosis factors but also by extracellular matrices (1).
BMPs are members of the TGF- β superfamily,
and their production and receptors have been detected in both epithelial and mesenchymal cells during tooth development (2, 3). Previous studies using the organ culture system and genetic mousemodels have demonstrated the critical role of BMPs
in tooth development and its morphogenesis (4, 5).
Moreover, two BMP antagonists, follistatin and ectodin (USAG - 1, Wise), have recently been reported
to control tooth morphogenesis by fine - tuning of
BMP signals (6 - 9). In follistatin transgenic mice,follistatin negatively regulated ameloblast differentiation and resulted in no enamel formation in theincisors (6). Conversely, follistatin knockout miceectopically produced enamel in the enamel free area
The Journal of Medical Investigation Vol. 55 2008

The Journal of Medical Investigation(the lingual side of incisors in rodents) (7). Ectodin has been shown to controls the activity of theenamel knot, a signal center, in tooth germ (10).
Consistently, loss of function analysis using ectodin knockout mice revealed both extra - tooth formation and altered cusp patterning (8, 9, 11, 12).
On the other hand, the application of BMP4 to theectodin - deficient tooth germ culture accelerated thecytodifferentiation of ameloblasts and odontoblasts(9). In addition, BMP2 has been also reported as
one of the potent inducers of ameloblast differentiation (amelogenesis) in combination with the apatite in vitro system (13). These results indicatedthat BMPs play an essential role in tooth development. However, the downstream molecular eventsand critical target genes of BMPs during amelogenesis remain still unclear.
HAT - 7 cells are dental epithelial cells derived
from the apical loop of rat incisors where is the dental stem cell niche (14). In addition, HAT - 7 cells
have a potential to differentiate into ameloblasts bycell - cell contact and overexpression of Notch signaling in vitro (15, 16).
In this study, we examined the regulatory rolesof BMP2 on gene expression in HAT-7 cells using
microarray and RT-PCR analysis.

MATERIALS & METHODS
Materials
Dulbecco’s Modified Eagle Medium (DMEM)
and Ham’s F - 12 Medium (F12) were purchasedfrom Nissui Pharmaceutical (Tokyo, Japan). Fetal
Bovine Serum (FBS) was purchased from Sigma Aldrich Corporation (St.Louis, MO, USA). BMP2
was provided by Yamanouchi Pharmaceutical(Tokyo, Japan). TRI Reagent was purchased from
Molecular Research Center, Inc. (Cincinnati, OH,
USA). RNA - PCR kit was from Takara Bio Inc.
(Otsu, Japan). Go Taq DNA polymerase was purchased from Promega (Madison, WI, USA) andSYBR Green PCR Master Mix was from Applied
Biosystems (Foster City, CA, USA).

Cells and cell culture
HAT - 7 cells (14) were maintained in DMEM/F
12 medium with 10% FBS under 5% CO2 in air at!. BMP2 was added when the cell growth was37!
observed at 80% confluence. After the cells were
washed twice with phosphate - buffered saline (PBS)
without calcium and magnesium [PBS (-) : 137 mM

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NaCl, 8.1 mM Na2HPO4 "12H2O, 2.68 mM KCl, and1.47 mM KH2PO4, pH 7.4], they were cultured withor without 300 ng/ml of BMP2.

Western blot analysis
HAT - 7 cells were lysed in a buffer containing
1% (v/v) Nonidet P - 40, 150 mM NaCl, 0.1% SDS,
1 mM EDTA, 10% glycerol, 10 mM NaF, 2 mM
phenylmethanesulfonyl fluoride, 0.1 mM leupeptin,
and 20 mg/ml aprotinin in 50 mM Tris-HCl (pH!. Lysates were sepa8.0) and rotated for 1 hr at 4!
rated by centrifugation at 20,000 !g for 15 min andsupernatants were collected in a microtube. Measurement of protein contents and Western blot analysis were carried out as described previously (17).
Activated Smad1/5/8 was detected using anti phospho - Smad1/5/8 (1 : 1,000 ; #9511, Cell Signaling, Danvers, MA, USA).

cDNA microarray analysis
HAT - 7 cells were cultured in DMEM/F12 medium with 10% FBS until 80% confluence was observed. The medium was then replaced by serumfree medium with or without 300 ng/ml of BMP2.
After 24 hrs, the cells were harvested and total RNA
was isolated using TRI Reagent followed by tworounds of ethanol precipitation. Each 20 μg of total
RNA was reverse transcribed and labeled with Cy3
or Cy5. The labeled probes were hybridized with
a cDNA microarray that printed 14,815 cDNA fragments (Agilent, Palo Alto, Cal., USA) on the slide.
The arrays were scanned and analyzed. Differential expression of the genes was determined aspreviously reported (17).

Quantitative RT-PCR
Total RNA was treated with DNase I (Invitrogen, Carlsbad, CA) to eliminate genomic DNA contamination. One μg of total RNA was reversetranscribed to cDNA using RNA - PCR kit following the manufacturer’s instruction. A part of thisreaction was used as a template in the polymerase
chain reaction (PCR) with SYBR Green PCR Master mix (Applied Biosystems) or FastStart Universal SYBR Green Master (ROX) (Roche Diagnostics, Mannheim, Germany). PCR amplification
was performed on the following genes ; Cyp26b,
CRP2, β-igh3, FN1, Smad7, OBF-1, TIEG , BMP2,
BMP4, BMP7, follistatin, ectodin and 18S rRNA. The
primer sequences are listed in Table 1. The ABIPRISM 7000 (Applied Biosystems) was used for
Quantative RT - PCR and expression ratios were cal-

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K. Miyoshi, et al. Effects of BMP2 on gene expressions in HAT-7

Table 1 Primers for Quantative RT-PCR
Gene
18S

Primers
5’- TACCTGGTTGATCCTGCCAGTAGCAT - 3’
5’- CCCGTCGGCATGTATTAGCTCTAGAA - 3’
Amelogenin 5’- CAAGAAATGGGGACCTGGATCTTGTTT - 3’
5’- GCTGCCTTATCATGCTCTGG - 3’
Cyp26b 5’- AGGGCCTAGAGAAGGCTGTC - 3’
5’- GTTCCTCCCGTAGCTTTTCC - 3’
Crp2
5’- GATCATTGGAGCTGGAAAGC - 3’
5’- TCCTTGACCATAGCCGAATC - 3’β- igh3
5’- CATTGACGGCCAAATGAAGAC - 3’
5’- TGGTGAACAGGGTCCCATAC - 3’
Fibronectin 5’- GAAAGGCAACCAGCAGAGTC - 3’
5’- CTGGAGTCAAGCCAGACAC - 3’
Smad7
5’- TTGGAGTCCTTTCCTCTCTC - 3’
5’- GGCTCAATGAGCATGCTCAC - 3’
OBF - 1 5’- CTGTCAGCTGCCCTTACTCC - 3’
5’- GGCCACGGGAAATAGGTAAG - 3’
TIEG
5’- ATCTGTAGCCACCCAGGATG - 3’
5’- GGGACAGGCAAATTTCTTCTC - 3’
BMP2
5’- TGAACACAGCTGGTCTCAGG - 3’
5’- GCTAAGCTCAGTGGGGACAC - 3’
BMP4
5’- CAGAGCCAACACTGTGAGG - 3’
5’- TCCACTCCCTTGAGGTAACG - 3’
BMP7
5’- AGACGCCAAAGAACCAAGAG - 3’
5’- GCTGTCGTCGAAGTAGAGGA - 3’
Follistatin 5’- CGGAACCCTCATCTTCAGAG - 3’
5’- GCCAACCTTGAAATCCCATA - 3’
Ectodin 5’- GAGGCAGGCATTTCAGTAGC - 3’
5’- CATAGCCTCCTCCGATCCAG - 3’

culated from the threshold cycles, normalized by
18S rRNA results. All data were confirmed by triplicate experiments.

RT-PCR analysis
Total RNA was treated with DNase I (Invitrogen, Carlsbad, CA) to eliminate genomic DNA contamination. One μg of the treated RNA was converted to cDNA in a 20 μl using RNA - PCR kit. One
μl of this reaction was used as a template in the PCR
with Go Taq DNA polymerase. The primer sequences are listed in Table 1 and 2. The results

Table 2 Primers for RT-PCR
Gene
GAPDH

Primers
5’- CATTGACCTCAACTACATGG - 3’
5’- CTCAGTGTAGCCCAGGATGC - 3’
P75Ngfr
5’- GAGGGCACATACTCAGACGAAGCC - 3’
5’- GTCTATATGTTCAGGCTGGTAACC - 3’
Jagged1
5’- GTCCACGGCACCTGCAATG - 3’
5’- ACAAGGCTTGGCCTCGCAC - 3’
Amelogenin 5’- CAAGAAATGGGGACCTGGATC - 3’
5’- GCTGCCTTATCATGCTCTGG - 3’Notch15’- CTACAATCAGGGCACCTGTG - 3’
5’- GCCATCACTGAAGTGGTCC - 3’
5’- CTGGACCTCATCAGCATTTG - 3’
Alkaline
phosphatase 5’- GCTGTGAAGGGCTTCTTGTC - 3’
Hes1
5’- CACGCTCGGGTCTGTGCTGAGAGC - 3’
5’- ATGCCAGCTGATATAATGGAG - 3’Notch25’- GAGTGTCTGAAGGGCTACGC - 3’
5’- TGCAGATGCAGGTGTAGGAG - 3’

were captured using Chemi - Doc XRS (BIO - RAD,Tokyo, Japan) and expression levels were estimatedusing Quantity One software (BIO - RAD).

RESULTS
BMP2-Smads signaling is functional in the dental
epithelial cell line, HAT-7
Previously, Coin, et al . reported that BMP2 combined with apatite could induce both morphological and functional differentiation of ameloblasts using the in vitro culture system (13). However, theprecise molecular mechanism of ameloblast differentiation remains unclear. To analyze the roles of
BMP2 on ameloblast differentiation, we first examined whether the BMP2 - Smads pathway is functional in HAT - 7 cells. Western blot analysis using
the antibody against the phosphorylated Smad1/5/8 demonstrated that BMP2 specifically activatedSmad1/5/8 within 15 min and maintained theirphosphorylated state up to 24 hrs in HAT - 7 cells
as shown in Fig. 1.

Fig. 1 Smad1/5/8 activation by BMP2 in HAT-7 cells.
HAT-7 cells were treated with 300ng/ml of BMP2 during the indicated time. Forty μg of total protein was applied in each lane.
Phospho-Smad1/5/8 was detected. Beta-actin was used as the internal control.

The Journal of Medical Investigation

Gene profiling in BMP2-treated HAT-7cells
To examine the effects of BMP2 on gene expression patterns in HAT - 7 cells, we performed cDNA

Table 3 Up-regulated genes by BMP2 in HAT-7
Gene Name
Fold
Rat cytochrome P450RAI-2 (Cyp26b1)
14.31
Rat cysteine and glycine-rich protein 2
6.68
Rat insulin-like growth factor binding protein 5
6.44
Rat rho GTPase activating protein 7
6.14
Rat beta ig-h3
5.87
Rat synaptotagmin-like 2
5.24
Rat DOC-2 p82
4.88
Rat carbonic anhydrase III (CA3)
4.85
Mouse capping protein alpha 1
4.19
Rat mRNA for fibronectin
4.06
Mouse NCAM-140 and NCAM-180 isoforms.
3.91
Mouse putative membrane-associated guanylate kinase
3.75
1 (Magi-1)
Mouse cytohesin binding protein (Cbp)
3.72
Rat lysyl oxidase
3.64
Rat Smad7 protein
3.50
Rat secernin 1
3.41
Rat RDC-1 protein.
3.16
Rat protein kinase C epsilon subspecies.
3.13
Mouse pre B-cell leukemia transcription factor 1
3.11
Rat New England Deaconess transcription factor
3.08
Rat G-protein beta 5 subunit
2.86
Mouse Id4 dominant negative helix - loop - helix gene. 2.74
Rat mRNA for gal beta 1,3 galNAc alpha 2,3- sialyltrans2.73
ferase.
Connective tissue growth factor precursor
2.70
Rat podocalyxin
2.69
Rat adenylyl cyclase
2.68
Plakophilin 2a2.66
Periostin, osteoblast specific factor
2.63
Mouse nicotinamide nucleotide transhydrogenase (Nnt)
2.62
Mouse TGF-beta-inducible protein (TSC-36)
2.61
Rat low voltage -activated, T-type calcium channel alpha2.56
subunit (CACNA1G)
Rat PGES mRNA for prostaglandin E synthase
2.56
Rat activin type I receptor
2.55
Rat tensin1 (Tns)
2.53
Mouse hereditary multiple exostoses (Ext1)
2.43
Rat peroxisome proliferator-activated receptor gamma
2.42
2 (PPARgamma2)
Rat mindin precursor,
2.42
Rat developmentally regulated intestinal protein (OCI-5) 2.39
Rat mRNA for multidrug resistance protein (MRP5)
2.33
Rat alpha 1 type V collagen
2.29
Rat follistatin-related protein precursor
2.28
Actin binding LIM protein family, member 3
2.25
Mouse homeobox gene Prx2 mRNA.
2.25
Mouse HIC-5
2.18
Rat lung beta-galactoside-binding lectin
2.15
Rat alpha-tropomyosin
2.05
Mouse Ulip protein.
2.03
Mouse sialic acid binding Ig-like lectin 10
2.03

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microarray analysis. Before the experiment, we
checked the effects of BMP2 on the amelogenin
gene expression and found that BMP2 could induce amelogenin expression transiently in dose and time - dependent manner (data not shown).
Based on the results, HAT - 7 cells were treated withor without 300 ng/ml of BMP2 for 24 hrs, and total
RNA was purified as mentioned in MATERIALS& METHODS. We found that 101 genes were regulated in response to BMP2 treatment (Tables 3 and
4). Seventy - three genes were up - regulated including 25 unknown genes and 28 genes were down regulated including 8 unknown genes (Fig. 2A).
The results were summarized based on the genefunction in Fig. 2B. Interestingly, 18 % of BMP2induced genes are ECM/adhesion molecules andsignaling molecules. Gene expressions of this category including fibronectin (18), NCAM (19), andperiostin (20, 21) have been reported in both ectomesenchyme and dental epithelia during toothdevelopment. About 10% of BMP2 - induced genes
are transcriptional regulators including Prx2, Id4,
PBX , and CRP2. However, their roles in tooth development have not been described in detail.

Confirmation of gene expression by Quantative
RT-PCR
To confirm the microarray data, we further ana-

Table 4 Down-regulated genes by BMP2 in HAT-7
Gene Name
Fold
Mouse C57Bl/6J clone L45 odorant receptor
0.48
Rat transcription factor Maf1
0.48
Kruppel-like factor 10, (TIEG)
0.47
Rat cDNA for glutamate receptor subunit (GluR6), kai0.46
nate subtype.
Extensin-like protein
0.46
Rat mRNA for carbonic anhydrase II.
0.45
Rat brain glucose -transporter protein
0.45
Mouse Mort1 Fas-associated protein
0.42
Mouse hybrid receptor gp250 precursor
0.41
Mouse cytoplasmic protein Ndr1 (Ndr1)
0.41
Rat high affinity glutamate transporter EAAC1
0.39
Mouse neural precursor cell expressed developmentally
0.39
downregulated Nedd9 (Nedd9)
Mouse Oct binding factor 1 (OBF-1)
0.39
Rat thrombomodulin
0.38
Incyte EST/inositol 1,4,5-trisphosphate 3-kinase C
0.37
Rat GADD153
0.35
Rat galectin-7
0.31
Rat PFK-L mRNA for liver phosphofructokinase.
0.27
Rat aldehyde dehydrogenase
0.25
Rat interferon regulatory factor 1 (IRF-1)
0.21

220
A
Up
Down
Total

B

K. Miyoshi, et al. Effects of BMP2 on gene expressions in HAT-7
Known
Unknown
Total
Gene
Gene
48
25
73
20
8
28
68
33
101
(more than 2-fold, less than 0.5 -fold.)

A[UP]
Secretion :Growth factor (1)Secreting machinery (2)Cell-cell interaction :ECM/Adhesion (13)Cell surface : Receptor (membrane) (2)Transporter/Channel (2)Membrane protein (1)Cytoskeleton : Cytoskeleton (2)Cytosol :Kinase (2)Enzyme (w/o kinase) (7)Signaling (8)Nuclear :Transcriptional regulator (8)[DOWN]
Apoptosis :Apoptosis (2)Cell-cell interaction :Adhesion/Glycoprotein (3)Cell surface : Receptor (membrane) (3)Transporter (2)Cytoskeleton : Cytoskeleton (1)Cytosol :Kinase (3)Enzyme (w/o kinase) (2)Signaling (1)Nuclear :Transcriptional regulator (3)Fig. 2 Summary of microarray analysis.
(A) Overview of gene expression change. “Up” and “Down”
indicate that the expression level is 2-fold higher or less than
0.5-fold by BMP2 treatment. (B) Functional classification of
up- and down-regulated genes. The number in parenthesis indicates the gene numbers.

lyzed the following 7 genes involved in developmental events : Cytochrome P450 family26 subfamily b (Cyp26b) (22), cysteine and glycine-rich protein
2 (CRP2) (23, 24), TGFβ-induced gene H3 (βig-h3)(25), Fibronectin 1 (FN1) (18, 26), Smad7 (27), Octbinding factor-1 (OBF ) (28, 29), and TGFβ-inducible
early transcription factor gene (TIEG/KLF10) (30)
(Fig. 3A). In silico analysis of the potential BMP inducible Smads - binding sites located within the
3.0 kb upstream region from each transcription startsite identified that the numbers of binding sites in
Cyp26b, CRP2, βig - h3, FN1, Smad7, OBF , and
TIEG were 79, 69, 59, 55, 92, 54, and 79, respectively. Subsequently, we performed the quantitative
RT - PCR to validate the microarray results as shownin Fig. 3B. These results indicated that the microarray data are reproducible. To further examine these
responses, we also performed RT - PCR analysis
using additional two other dental epithelial cell lines
treated with BMP2. The result shows similar responses to BMP2 treatment as HAT - 7 cells (dataB

Fig. 3 Confirmation of microarray data by Quantative RT-PCR.
(A) The changes in the expression levels of genes related to differentiation. Up- and down-regulated genes are indicated by upand down-arrows, respectively. (B) Confirmation of microarray data by Quantative RT-PCR. Expression levels of each geneare normalized to those of 18S rRNA. Relative level is calculated as BMP2 treatment sample divided by untreated sample.
White bar : untreated HAT-7 as a control, striped bar : 300 ng/
ml of BMP2 treated HAT-7.

not shown).

BMP2 enhanced gene expression of ameloblast
markers in HAT-7cells
Since the number of cDNA that can be printed
on a microarray is limited, we could not detect any
well-known amelogenesis-related genes. Dental
epithelial cells differentiate into four cell-lineages
during tooth development : inner enamel epithelium(IEE), stratum intermedium (SI), stratum reticulum (SR), and outer enamel epithelium (OEE).
Ameloblasts are derived from IEE by several growth
factors, cytokines, and cell - cell interactions. To assess whether BMP2 can promote in vitro differentiation of undifferentiated HAT-7 cells into inner
enamel epithelial - lineage cells or ameloblasts, weanalyzed mRNA induction of marker genes following BMP2 treatment.
Fig. 4A depicts well - known differentiation markers related to the dental epithelial cell - lineages (16).
As shown in Fig. 4B, BMP2 enhanced the gene expressions of p75Ngfr and amelogenin (IEE andameloblast markers) whereas that of Hes1 (SImarker) was slightly reduced. Moreover, the gene
expression of Notch2 (SR or OEE marker) was
also enhanced by BMP2 treatment. The othermarker genes for IEE and SI, Notch1 and Jagged 1,

The Journal of Medical Investigation

were unaffected. These results indicated that BMP2
could promote ameloblastic marker genes induction.
Furthermore, we analyzed the effects of BMP2
on the gene expression of other BMP families andtheir antagonists that are closely related to amelo-Vol. 55 August 2008

221

genesis. BMP2 treatment enhanced the expression level of BMP4 (Fig. 5A, 5B), and significantly
induced that of ectodin in HAT - 7 cells (Fig. 5A).
Conversely, the expression levels of BMP2, BMP7,
and follistatin were unaffected by BMP2 treatment(Fig. 5A, 5B).

A

B

Fig. 4 Effects of BMP2 on marker gene expression of four dental epithelial cell-lineages in HAT-7 cells.
(A) The localization of four dental epithelial cell-lineages at bell stage of tooth germ. The marker genes of each cell-lineage are indicated under the name of the cell-lineage.
IEE : inner enamel epithelium, SI : stratum intermedium, SR : stratum reticulum, and OEE : outer enamel epithelium. (B) Effects of
BMP2 on 4 -cell-lineage marker genes in HAT-7 cells. Left panel : RT-PCR analysis was performed and PCR products were stained
and captured using Chemi-Doc XRS. C : no treatment, B2 : 300ng/ml BMP2 was treated for 24 hrs. Right panel : relative ratio ofgene expression levels. The expression levels were estimated using Quantity One software, and normalized by each 18S rRNA level.
Relative level was calculated as BMP2 treatment sample divided by untreated sample. IEE : inner enamel epithelium, SI : stratum
intermedium, SR : stratum reticulum, OEE : outer enamel epithelium, p75 : p75Ngfr, J1 : Jagged1, AG : amelogenin, N1 : Notch1,
ALP : alkaline phosphatase, H1 : Hes1, N2 : Notch2. White bar : untreated HAT-7 as a control, striped bar : 300 ng/ml of BMP2
treated HAT-7. These are representative data among three repeated experiments.

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K. Miyoshi, et al. Effects of BMP2 on gene expressions in HAT-7

A

B

Fig. 5 Effects of BMP2 on BMP2, 4, 7 and BMP antagonists,
ectodin, follistatin mRNAs in HAT-7 cells.
(A) RT-PCR analysis was performed. The ectodin mRNA could
not be detected initially, but it was induced by BMP2 in HAT-7
cells. C : untreated, B2 : BMP2 treated with 300ng/ml for 24
hrs. (B) The relative level of each gene expression in HAT-7
cells. Quantitative RT-PCR was performed on BMP2, 4, 7 and
BMP antagonists, ectodin, follistatin mRNAs in HAT-7 cells.
Each expression level was normalized by the 18S rRNA level.
Relative level was calculated as BMP2 treated sample divided
by untreated sample. White bar : untreated HAT-7 as a control,
striped bar : 300 ng/ml of BMP2 treated HAT-7 cells. PCR products of Ectodin were not detected in untreated HAT-7 cells.
Therefore, the relative ratio could not be calculated.

DISCUSSION
Here we reported that canonical BMP - Smad signaling pathway in HAT-7 cells could be activatedin response to BMP2 stimulation (Fig. 1). Moreover, BMP2 was able to enhance the expression
levels of p75Ngfr and amelogenin, the well-known
ameloblast differentiation markers, whereas that of
Hes1, a marker for stratum intermedium, was reduced (Fig. 4). These findings suggest that the gene
expression pattern in HAT-7 cells was shifted fromthe transient amplifying stage of IEE (15) to theameloblast - lineage by BMP2 treatment which isconsistent with the results of the previous study sug-gesting that BMP may play a role in amelogenesis(13). The other marker genes for IEE and SI, Jagged1, Notch1, and ALP were unaffected. We needfurther investigation for these genes but it is possible that the effects of BMP2 are not so significant
on these gene expressions at this time point. Recently, it has been reported that Jagged1 is one of
the target genes of Shh signaling (31), and Notch1
is regulated via IKK signaling (32). Furthermore,
the induction of ALP gene expression is mediatedthrough MAPK activity (33), and Wnt signaling suppresses BMP2 - induced ALP expression in osteoblasts (34). These data suggested that othersignaling molecules might be involved in the regulation of their gene expression.
Taken together, we conclude that HAT-7 cells area suitable in vitro assay system to analyze BMP2 mediated gene regulation.
We performed microarray analysis using total
RNA from BMP2 - treated and untreated HAT - 7
cells. The results clearly demonstrated that BMP2
regulated multiple gene expression positively andnegatively in HAT - 7 cells as listed in Fig. 2B. Up regulated genes are listed in Table 3 and include
several tooth - related genes. Connective tissue growth
factor precursor was up - regulated 2.7 - fold by BMP2
in HAT - 7 cell, and the expression of connective tissue growth factor (CTGF ) has been reported in IEE,
preameloblast, and OEE (35). Periostin was up regulated 2.6 - fold by BMP2, and it has been detected in SI cells (21). Notably, periostin null mice
showed abnormal ameloblast differentiation (20).
To further analyze the microarray data, we focused on 7 genes that are reported to play a role incellular differentiation. The highest induction wasobserved with Cyp26b1 gene expression in BMP2
treated HAT - 7 cells as shown in Table 3, Cyp26b1,
belonging to the Cyp26 family are a group of P450
enzymes that metabolize retinoic acid to inactive
forms (36). Cyp26b1 is highly expressed in the restricted regions of the developing limb, and a knockout mouse model showed that Cyp26b1 prevents
apoptosis and promotes chondrocyte maturation inlimb development (37). The Cyp26 gene has two
additional isoforms, Cyp26a1 and Cyp26c1, thathave been reported to be expressed in mouse dental epithelium (22, 38, 39) ; Cyp26b1 has been detected in the craniofacial mesenchymal cells neighboring Cyp26a1- and Cyp26c1-expressing cells (22,39). Fibronectin (FN ), an extracellular matrix protein, is also induced by BMP2. In tooth development, FN is expressed not only in mesenchymal

The Journal of Medical Investigation

cells but also in the stellate reticulum and is deposited in basement membrane (18, 40). FN has also
been shown to appear in the epithelium during submandibular gland development and is suggested toinduce the EMT event in development of the cleftof alveoli (26). Since HAT - 7 cells are derived froman apical loop containing stem cell fraction, it ispossible that some population of HAT - 7 cells could
differentiate into SR cells. Smad7, an inhibitory
Smad, was increased 3.5 - 4 - fold in BMP2 treated
HAT - 7. Smad7 has been shown to inhibit TGF - βsignaling to a greater degree than BMP signaling(41, 42). It has also been reported that keratin 5
promoter - driven Smad7 transgenic mice caused
defects in amelogenesis (27). These results suggested that BMP2 may define the spatio-temporal
domain of BMP target field by balancing between
TGF - β and BMP signaling through regulating
Smad7 gene expression. OBF is a B - cell specifictranscriptional co - activator that functions with octamer binding transcription factors to regulate B
cell differentiation (28). TIEG (KLF10) was identified as a TGF - β inducible gene and it was also reported that BMP2 could induce TIEG mRNA andregulate cerebellar granule cell differentiation (43,44). However, OBF and TIEG were down - regulated
in BMP2 - treated HAT - 7 and this may be due to thedifference in cell types (Table 4 and Fig. 3).
Moreover, we discovered two interesting genesthat have never been discussed in amelogenesis
study. One is CRP2, a member of the LIM familyof proteins, which contains the LIM motif and double zinc finger domains (24). In general, LIM proteins are important for both cellular differentiationand function (45). LIM family proteins, such as Islet1, Lhx6, Lhx7, and Lhx8, have shown to be important regulators in tooth formation and patterning (46). Islet1 is expressed in dental epithelium
and activates BMP4 expression. Lhx 6, 7, and 8 are
expressed in dental mesenchyme (47). CRP2 hasbeen reported to have restricted expression in arteries, cardiac myocytes, and fibroblasts (24, 48) ;however, there are no reports of its expression in
tooth development. Our microarray data indicatedthat BMP2 strongly induces CRP2 mRNA in thedental epithelial cell line (6.7 - fold compare to untreated HAT-7 cells). Western blot analysis could
also detect enhanced CRP2 protein expression in
BMP2 - treated HAT - 7 cells (unpublished results).
However, we could not detect specific CRP2 reactivity in the rat tooth during amelogenesis by immunohistochemistry (data not shown). Therefore,

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further investigation is required to determine theexpression of CRP2 in tooth structures and to analyze its role in amelogenesis.
Another gene identified in this study is βig - h3,
originally recognized as a TGF - β inducible gene,whose mRNA was increased 5.9 - fold by BMP2
treatment. βig - h3 is a secretory protein and a member of the periostin family (49) that contains bothan RGD sequence and four fasciclin - 1 domains. It
is expressed in several tissues, including lung, kidney, skin, bone, and cartilage (25, 50, 51). βig - h3
has been shown to bind not only to ECM proteinssuch as FN, laminin, and collagens, but also to cellsurface molecules such as αvβ5 integrin, decorin,and biglycan (52, 53). To confirm βig - h3 expression, we performed immunohistochemical analysis using a commercially available antibody. Unfortunately, we could not detect any specific signals in tooth sections, possibly due to the inabilityof the antibody to detect the antigen on paraffinembedded samples (data not shown). To understand the roles of CRP2 and βig - h3 in tooth development in vivo, it will be necessary to generate newantibodies against them and the gene knock - outmice in the next study.
Microarray data did not include other tooth- related genes such as BMP family members (BMP-2,
BMP-4, and BMP-7) and BMP antagonists (ectodin, follistatin) since the gene numbers on the microarray chip were limited. Therefore, we further
performed RT - PCR analysis to examine their expression as shown in Fig. 5. BMP2 treatment significantly enhanced the level of BMP4 mRNA. Thisfinding is novel and important in the dental epithelial cells since BMP4 plays a distinct role in early
tooth development (5). Moreover, RT - PCR analysis demonstrated that BMP2 regulated the expression of its own antagonists. BMP2 induced ectodinmRNA expression and slightly enhanced follistatinmRNA expression (Fig. 5B). Since ectodin is also
an inhibitor of Wnt via competitive binding to LRP
6 (54), both BMP and Wnt might form a regulatory loop in vivo. Previous studies demonstrated
that both ectodin and follistatin are expressed in
IEE and OEE, and play important roles in toothmorphogenesis (6 - 9). Since overexpression of
Wnt3 caused defects of ameloblast differentiationin the lower incisor (55), it is suggested that theinduction of ectodin may form a negative feedback
loop serving to control ameloblast differentiation
in HAT - 7 cells.
Taken together, our findings suggest that BMP2

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K. Miyoshi, et al. Effects of BMP2 on gene expressions in HAT-7

might accelerate or support amelogenesis at themolecular level. Therefore, our gene profiling data
will facilitate identification of a new approach forthe molecular analysis of amelogenesis and tooth
regeneration.

8.

ACKNOWLEDGMENTS
This work was partly supported by Grants - in Aid for scientific research (No. 17689051) from the
Ministry of Education, Culture, Sports, Science andTechnology, Japan. We thank Ms. Mami Sawatarifor technical assistance.

9.

10.

REFERENCES
1.

2.

3.

4.

5.

6.

7.

Thesleff I : Epithelial - mesenchymal signalling
regulating tooth morphogenesis. J Cell Sci 116 :1647 - 8, 2003
Aberg T, Wozney J, Thesleff I : Expression patterns of bone morphogenetic proteins (Bmps)
in the developing mouse tooth suggest roles inmorphogenesis and cell differentiation. Dev
Dyn 210 : 383 - 96, 1997
Nadiri A, Kuchler - Bopp S, Perrin - Schmitt F,
Lesot H : Expression patterns of BMPRs in thedeveloping mouse molar. Cell Tissue Res 324 :
33 - 40, 2006
Andl T, Ahn K, Kairo A, Chu EY, Wine - Lee L,
Reddy ST, Croft NJ, Cebra-Thomas JA, Metzger
D, Chambon P, Lyons KM, Mishina Y, Seykora
JT, Crenshaw EB, 3rd, Millar SE : Epithelial
Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development. Development 131 :
2257 - 68, 2004
Bei M, Kratochwil K, Maas RL : BMP4 rescues
a non - cell - autonomous function of Msx1 in
tooth development. Development 127 : 4711 - 8,
2000
Wang XP, Suomalainen M, Jorgez CJ, Matzuk
MM, Werner S, Thesleff I : Follistatin regulates enamel patterning in mouse incisors byasymmetrically inhibiting BMP signaling andameloblast differentiation. Dev Cell 7 : 719 - 30,
2004
Wang XP, Suomalainen M, Jorgez CJ, Matzuk
MM, Wankell M, Werner S, Thesleff I : Modulation of activin/bone morphogenetic protein
signaling by follistatin is required for the mor-

11.

12.

13.

14.

15.

16.

phogenesis of mouse molar teeth. Dev Dyn
231 : 98 - 108, 2004
Murashima-Suginami A, Takahashi K, Kawabata
T, Sakata T, Tsukamoto H, Sugai M, Yanagita
M, Shimizu A, Sakurai T, Slavkin HC, Bessho
K : Rudiment incisors survive and erupt as supernumerary teeth as a result of USAG - 1 abrogation. Biochem Biophys Res Commun 359 :
549 - 55, 2007
Kassai Y, Munne P, Hotta Y, Penttila E,
Kavanagh K, Ohbayashi N, Takada S, Thesleff
I, Jernvall J, Itoh N : Regulation of mammalian tooth cusp patterning by ectodin. Science
309 : 2067 - 70, 2005
Laurikkala J, Kassai Y, Pakkasjarvi L, Thesleff
I, Itoh N : Identification of a secreted BMP antagonist, ectodin, integrating BMP, FGF, andSHH signals from the tooth enamel knot. Dev
Biol 264 : 91 - 105, 2003
Yanagita M, Oka M, Watabe T, Iguchi H,
Niida A, Takahashi S, Akiyama T, Miyazono
K, Yanagisawa M, Sakurai T : USAG -1 : a bonemorphogenetic protein antagonist abundantly
expressed in the kidney. Biochem Biophys
Res Commun 316 : 490 - 500, 2004
Yanagita M, Okuda T, Endo S, Tanaka M,
Takahashi K, Sugiyama F, Kunita S, Takahashi
S, Fukatsu A, Yanagisawa M, Kita T, Sakurai
T : Uterine sensitization - associated gene - 1(USAG - 1), a novel BMP antagonist expressedin the kidney, accelerates tubular injury. J Clin
Invest 116 : 70 - 9, 2006
Coin R, Haikel Y, Ruch JV : Effects of apatite,transforming growth factor beta - 1, bone morphogenetic protein - 2 and interleukin - 7 on
ameloblast differentiation in vitro. Eur J OralSci 107 : 487 - 95, 1999
Kawano S, Morotomi T, Toyono T, Nakamura
N, Uchida T, Ohishi M, Toyoshima K, Harada
H : Establishment of dental epithelial cell line
(HAT - 7) and the cell differentiation dependent
on Notch signaling pathway. Connect Tissue
Res 43 : 409 - 12, 2002
Harada H, Ichimori Y, Yokohama-Tamaki T,
Ohshima H, Kawano S, Katsube K, Wakisaka
S : Stratum intermedium lineage diverges fromameloblast lineage via Notch signaling. Biochem Biophys Res Commun 340 : 611 - 6, 2006
Kawano S, Saito M, Handa K, Morotomi T,
Toyono T, Seta Y, Nakamura N, Uchida T,
Toyoshima K, Ohishi M, Harada H : Characterization of dental epithelial progenitor cells

The Journal of Medical Investigation

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

derived from cervical - loop epithelium in a rat
lower incisor. J Dent Res 83 : 129 - 33, 2004
Ruspita I, Miyoshi K, Muto T, Abe K, Horiguchi
T, Noma T : Sp6 downregulation of follistatin
gene expression in ameloblasts. J Med Invest
55 : 87 - 98, 2008
Thesleff I, Barrach HJ, Foidart JM, Vaheri A,
Pratt RM, Martin GR : Changes in the distribution of type IV collagen, laminin, proteoglycan, and fibronectin during mouse tooth development. Dev Biol 81 : 182 - 92, 1981
Obara N, Suzuki Y, Nagai Y, Nishiyama H,
Mizoguchi I, Takeda M : Expression of neuralcell - adhesion molecule mRNA during mouse
molar tooth development. Arch Oral Biol 47 :805 - 13, 2002
Kii I, Amizuka N, Minqi L, Kitajima S, Saga Y,
Kudo A : Periostin is an extracellular matrix
protein required for eruption of incisors inmice. Biochem Biophys Res Commun 342 :
766 - 72, 2006
Suzuki H, Amizuka N, Kii I, Kawano Y,
Nozawa - Inoue K, Suzuki A, Yoshie H, Kudo
A, Maeda T : Immunohistochemical localization of periostin in tooth and its surrounding
tissues in mouse mandibles during development. Anat Rec A Discov Mol Cell Evol Biol
281 : 1264 - 75, 2004
MacLean G, Abu - Abed S, Dolle P, Tahayato
A, Chambon P, Petkovich M : Cloning of anovel retinoic - acid metabolizing cytochrome
P450, Cyp26B1, and comparative expression
analysis with Cyp26A1 during early murine
development. Mech Dev 107 : 195 - 201, 2001
Herrmann J, Borkham - Kamphorst E, Haas U,
Van de Leur E, Fraga MF, Esteller M, Gressner
AM, Weiskirchen R : The expression of CSRP2
encoding the LIM domain protein CRP2 ismediated by TGF-beta in smooth muscle andhepatic stellate cells. Biochem Biophys Res
Commun 345 : 1526 - 35, 2006
Weiskirchen R, Gunther K : The CRP/MLP/
TLP family of LIM domain proteins : acting byconnecting. Bioessays 25 : 152 - 62, 2003
Ferguson JW, Mikesh MF, Wheeler EF,
LeBaron RG : Developmental expression patterns of Beta - ig (betaIG - H3) and its function
as a cell adhesion protein. Mech Dev 120 : 851 64, 2003
Sakai T, Larsen M, Yamada KM : Fibronectin
requirement in branching morphogenesis. Nature 423 : 876 - 81, 2003

Vol. 55 August 2008

225

27. Klopcic B, Maass T, Meyer E, Lehr HA,
Metzger D, Chambon P, Mann A, Blessing M :TGF - beta superfamily signaling is essential
for tooth and hair morphogenesis and differentiation. Eur J Cell Biol 86 : 781 - 99, 2007
28. Corcoran LM, Hasbold J, Dietrich W,
Hawkins E, Kallies A, Nutt SL, Tarlinton DM,
Matthias P, Hodgkin PD : Differential requirement for OBF - 1 during antibody - secreting celldifferentiation. J Exp Med 201 : 1385 - 96, 2005
29. Hess J, Nielsen PJ, Fischer KD, Bujard H,
Wirth T : The B lymphocyte - specific coactivator BOB.1/OBF.1 is required at multiple stagesof B-cell development. Mol Cell Biol 21 : 15319, 2001
30. Subramaniam M, Gorny G, Johnsen SA,
Monroe DG, Evans GL, Fraser DG, Rickard
DJ, Rasmussen K, van Deursen JM, Turner
RT, Oursler MJ, Spelsberg TC : TIEG1 nullmouse - derived osteoblasts are defective inmineralization and in support of osteoclast differentiation in vitro. Mol Cell Biol 25 : 1191 - 9,2005
31. McGlinn E, van Bueren KL, Fiorenza S, Mo
R, Poh AM, Forrest A, Soares MB, Bonaldo
Mde F, Grimmond S, Hui CC, Wainwright B,
Wicking C : Pax9 and Jagged1 act downstreamof Gli3 in vertebrate limb development. Mech
Dev 122 : 1218 - 33, 2005
32. Ohazama A, Hu Y, Schmidt - Ullrich R, Cao Y,
Scheidereit C, Karin M, Sharpe PT : A dualrole for Ikk alpha in tooth development. Dev
Cell 6 : 219 - 27, 2004
33. Jadlowiec J, Koch H, Zhang X, Campbell PG,
Seyedain M, Sfeir C : Phosphophoryn regulates
the gene expression and differentiation of
NIH3T3, MC3T3 - E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway. J Biol Chem 279 : 53323 - 30, 2004
34. Fujita K, Janz S : Attenuation of WNT signaling by DKK - 1 and - 2 regulates BMP2 - induced
osteoblast differentiation and expression of
OPG, RANKL and M - CSF. Mol Cancer 6 : 71,
2007
35. Shimo T, Wu C, Billings PC, Piddington R,
Rosenbloom J, Pacifici M, Koyama E : Expression, gene regulation, and roles of Fisp12/
CTGF in developing tooth germs. Dev Dyn
224 : 267 - 78, 2002
36. Fujii H, Sato T, Kaneko S, Gotoh O, Fujii Kuriyama Y, Osawa K, Kato S, Hamada H :Metabolic inactivation of retinoic acid by a

226

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

K. Miyoshi, et al. Effects of BMP2 on gene expressions in HAT-7

novel P450 differentially expressed in developing mouse embryos. Embo J 16 : 4163 - 73, 1997
Yashiro K, Zhao X, Uehara M, Yamashita K,
Nishijima M, Nishino J, Saijoh Y, Sakai Y,
Hamada H : Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse
limb. Dev Cell 6 : 411 - 22, 2004
Tahayato A, Dolle P, Petkovich M : Cyp26C1
encodes a novel retinoic acid - metabolizing enzyme expressed in the hindbrain, inner ear,first branchial arch and tooth buds during
murine development. Gene Expr Patterns 3 :449 - 54, 2003
Abu - Abed S, MacLean G, Fraulob V, Chambon
P, Petkovich M, Dolle P : Differential expression of the retinoic acid - metabolizing enzymes
CYP26A1 and CYP26B1 during murine organogenesis. Mech Dev 110 : 173 - 7, 2002
Jowett AK, Kimber SJ, Ferguson MW : Immunofluorescent lectin binding patterns andglycoprotein co - localization in the developing
murine molar tooth. Arch Oral Biol 37 : 303 14, 1992
Nakao A, Afrakhte M, Moren A, Nakayama T,
Christian JL, Heuchel R, Itoh S, Kawabata M,
Heldin NE, Heldin CH, ten Dijke P : Identification of Smad7, a TGFbeta - inducible antagonist of TGF - beta signalling. Nature 389 : 6315, 1997
Nagarajan RP, Zhang J, Li W, Chen Y : Regulation of Smad7 promoter by direct association
with Smad3 and Smad4. J Biol Chem 274 :
33412 - 8, 1999
Hefferan TE, Subramaniam M, Khosla S, Riggs
BL, Spelsberg TC : Cytokine - specific inductionof the TGF - beta inducible early gene (TIEG) :regulation by specific members of the TGF beta family. J Cell Biochem 78 : 380 - 90, 2000
Alvarez-Rodriguez R, Barzi M, Berenguer J,
Pons S : Bone Morphogenetic Protein 2 Opposes Shh - mediated Proliferation in Cerebellar Granule Cells through a TIEG - 1 - based
Regulation of Nmyc. J Biol Chem 282 : 37170 80, 2007
Kadrmas JL, Beckerle MC : The LIM domain :from the cytoskeleton to the nucleus. Nat Rev
Mol Cell Biol 5 : 920 - 31, 2004
Tucker A, Sharpe P : The cutting - edge ofmammalian development ; how the embryo
makes teeth. Nat Rev Genet 5 : 499 - 508, 2004

47. Mitsiadis TA, Angeli I, James C, Lendahl U,
Sharpe PT : Role of Islet1 in the patterning ofmurine dentition. Development 130 : 4451 - 60,
2003
48. Chang DF, Belaguli NS, Chang J, Schwartz
RJ : LIM - only protein, CRP2, switched onsmooth muscle gene activity in adult cardiac
myocytes. Proc Natl Acad Sci USA 104 : 157 62, 2007
49. Litvin J, Zhu S, Norris R, Markwald R : Periostin family of proteins : therapeutic targets
for heart disease. Anat Rec A Discov Mol Cell
Evol Biol 287 : 1205 - 12, 2005
50. Skonier J, Neubauer M, Madisen L, Bennett
K, Plowman GD, Purchio AF : cDNA cloning
and sequence analysis of beta ig - h3, a novel
gene induced in a human adenocarcinoma cellline after treatment with transforming growth
factor - beta. DNA Cell Biol 11 : 511 - 22, 1992
51. Skonier J, Bennett K, Rothwell V, Kosowski S,
Plowman G, Wallace P, Edelhoff S, Disteche
C, Neubauer M, Marquardt H, Rodgers J,
Purchio AF : beta ig-h3 : a transforming growth
factor - beta - responsive gene encoding a secreted protein that inhibits cell attachment invitro and suppresses the growth of CHO cells
in nude mice. DNA Cell Biol 13 : 571 - 84, 1994
52. Kim JE, Jeong HW, Nam JO, Lee BH, Choi
JY, Park RW, Park JY, Kim IS : Identificationof motifs in the fasciclin domains of the transforming growth factor - beta - induced matrix
protein betaig - h3 that interact with the alphavbeta5 integrin. J Biol Chem 277 : 46159 65, 2002
53. Reinboth B, Thomas J, Hanssen E, Gibson
MA : Beta ig - h3 interacts directly with biglycan and decorin, promotes collagen VI aggregation, and participates in ternary complexing
with these macromolecules. J Biol Chem 281 :
7816 - 24, 2006
54. Itasaki N, Jones CM, Mercurio S, Rowe A,
Domingos PM, Smith JC, Krumlauf R : Wise,a context - dependent activator and inhibitor of
Wnt signalling. Development 130 : 4295 - 305,2003
55. Millar SE, Koyama E, Reddy ST, Andl T,
Gaddapara T, Piddington R, Gibson CW : Overand ectopic expression of Wnt3 causes progressive loss of ameloblasts in postnatal mouseincisor teeth. Connect Tissue Res 44 (Suppl 1) :124 - 9, 2003