antagonistic effects of smad2 versus smad7 are sensitive ... · 9/13/2001 · jbc m0-11424 the...

JBC M0-11424 The level of Smad expression and tooth development

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Antagonistic effects of Smad2 versus Smad7 are sensitive to their expressionlevel during tooth development

Yoshihiro Ito1, Jingsong Zhao1, Ali Mogharei1, Charles F. Shuler1, Michael Weinstein2,Chuxia Deng3, and Yang Chai1*

1. Center for Craniofacial Molecular Biology School of Dentistry University of SouthernCalifornia, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033

2. Department of Molecular Genetics, The Ohio State University 484 West 12th Avenue,Columbus, OH 43210-1292

3. Genetics of Development and Disease Branch, NIDDK, National Institute of Health,10/9N105, 10 Center Drive, Bethesda, MD 20892

Key words: tooth development, Smad2, Smad7 attenuation and heterozygous mutation, cellproliferation and apoptosis.

8 Figures and 1 Table

(*) Address correspondence to:Yang Chai, D.D.S., Ph.D.Center for Craniofacial Molecular BiologyUniversity of Southern California2250 Alcazar Street, CSA 103Los Angeles, California 90033

Tel. (323)442-3480Fax (323)442-2981e-mail: [email protected]

Running Title: The level of Smad expression and tooth development

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 13, 2001 as Manuscript M011424200 by guest on D

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ABSTRACT

Members of the transforming growth factor-β (TGF-β) superfamily regulate cell proliferation,

differentiation and apoptosis, controlling the development and maintenance of most tissues.

TGF-β signal is transmitted through the phosphorylation of Smad proteins by TGF-β receptor

serine/threonine kinase. During early tooth development, TGF-β inhibits proliferation of enamel

organ epithelial cells but the underlying molecular mechanisms are largely unknown. Here we

tested the hypothesis that antagonistic effects between Smad2 and Smad7 regulate TGF-β

signaling during tooth development. Attenuation of Smad2 gene expression resulted in

significant advancement of embryonic tooth development with increased proliferation of enamel

organ epithelial cells, while attenuation of Smad7 resulted in significant inhibition of embryonic

tooth development with increased apoptotic activity within enamel organ epithelium. These

findings suggest that different Smads may have differential activities in regulating TGF-β-

mediated cell proliferation and death. Furthermore, functional haploinsufficiency of Smad2, but

not Smad3, altered TGF-β-mediated tooth development. The results indicate that Smads are

critical factors in orchestrating TGF-β-mediated gene regulation during embryonic tooth

development. The effectiveness of TGF-β signaling is highly sensitive to the level of Smad gene

expression.

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INTRODUCTION

Embryonic development results from an ordered series of gene interactions, each in turn

designating individual cell type proliferation and differentiation. Central to the regulation of

embryonic development is the determination of time- and position-restricted signaling

instructions during morphogenesis of different phenotypes, with tooth formation being a classic

example. It is now evident that a hierarchy of growth factors and their downstream transcription

factors compose a conserved “morphogenetic code” − a set of rules common to processes that are

used repeatedly in different combinations to make functional organs (1, 2). Transforming growth

factor-β (TGF-β) ligands are expressed in a time- and tissue-specific manner and are important in

regulating the formation of tooth and Meckel’s cartilage (3). Smad proteins relay TGF-β

signaling from the cell membrane to the nucleus. Extracellular TGF-β signals are transduced via

membrane-bound TGF-β type II and type I receptors, which phosphorylate intracellular Smad2

and Smad3. Activated Smad molecules appear to regulate the expression of transcription factors

and affect the transcriptional status of target genes (4).

Different members of the Smad family have distinct signaling functions. Smad1, 2, 3, and 5

interact with and are phosphorylated by specific type I serine/threonine kinase receptors, and

thereby act in a pathway-restricted manner. In particular, Smad2 and Smad3 are phosphorylated

and translocated to the nucleus after stimulation by TGF-β (5-8) or activin (9), whereas Smad1

and 5 are activated following BMP stimulation (10-13). Smad2 and Smad3 are very similar in

their structures. It is possible that there may be some redundancy in the functional activity of

these two family members.

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The action of Smad4 differs from other members of the Smad family. After ligand stimulation

and phosphorylation of pathway-restricted Smads, Smad4 forms hetero-oligomers with pathway-

restricted Smads (14, 6, 15). In mammalian cells, Smad4 forms complexes with Smad2 and

Smad3 after activation by TGF-β or activin (9, 16, 14, 15), whereas, it forms complexes with

Smad1 and 5 after activation by BMP (14).

Smad6 and Smad7 diverge structurally from other members of the Smad family (17, 18, 8, 19),

they function as inhibitors of TGF-β, activin and BMP signaling. In particular, Smad7 associates

stably with the TGF-β receptor complex, but is not phosphorylated upon TGF-β stimulation.

Smad7 inhibits TGF-β-mediated phosphorylation of Smad2 and Smad3. Because transcription of

the inhibitory Smad gene is induced by stimulation of TGF-β (8, 20), inhibitory Smads may

produce autoregulatory negative feedback in the signal transduction of the TGF-β superfamily.

Previous studies have shown that TGF-β subtypes have specific functions during critical

epithelial-mesenchymal interactions related to the formation of tooth and Meckel’s cartilage (21,

3). In particular, TGF-β2 exerts a negative regulation on the proliferation of enamel organ

epithelial cells through a possible autocrine mechanism during early stages of tooth development

(22). However, regulation of intracellular TGF-β signaling during early craniofacial

morphogenesis has not been defined. Recent study has shown that the expression of Smad1 and

Smad2 are closely associated with critical stages of odontogenesis (23). More importantly,

targeted disruptions of Smad genes have revealed important biological functions of these

intracellular signaling molecules. But because of the early embryonic lethality associated with

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the Smad null mutation, such as Smad2-/-, it has not been possible to investigate the biological

function of TGF-β signaling Smad during organogenesis.

Here, we investigated the functional roles of Smad2 and Smad7 during embryonic tooth

development using mandibular explants as the experimental model. Our experiments provide the

first biological evidence that Smad2 versus Smad7 have important and opposite regulatory roles

during early tooth formation. Furthermore, different Smad molecules may contribute to the

diverse biological functions of TGF-β signaling in regulating cell proliferation and apoptosis.

The outcome of TGF-β signaling is sensitive to the level of Smad expression.

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MATERIALS AND METHODS

Organ Culture

Timed-pregnant Swiss-Webster or C57BL/6 mice were sacrificed on postcoital day 11 (E11).

E11 embryos (42-44 somite pairs) were staged according to the external developmental

characteristics as described by Theiler (24). The first branchial arch explants (eight per dish)

were cultured for periods up to 9 days according to the standard methods (3).

Competitive PCR

Primers were designed based on the murine Smad2, Smad3 and Smad7 cDNA sequences.

Primers for Smad2 were 5’- TCA CAG TCA TCA TGA GCT CAA GG-3’ and 5’- TGT GAC

GCA TGG AAG GTC TCT C-3’. The PCR product size was 471 bp. Primers for Smad3 were

5’-GAG TAG AGA CGC CAG TTC TAC C-3’ and 5’-GGT TTG GAG AAC CTG CGT CCA

T-3’. The PCR product size was 234 bp. Primers for Smad7 were 5’-AAT GGC TTT TGC CTC

GGA CAG C-3’ and 5’-CAC AAA GCT GAT CTG CAC GGT G-3’. The product size was 321

bp. Construction of Smad2, 3 and 7 competitors was done as previously described by Zhao et al.

(25, 26).

In-situ hybridization

A 256 bp fragment of murine Smad2 cDNA subcloned into pBluescript II SK was digested

with Not I and transcribed with T7 RNA polymerase (Boehringer Mannheim) for an antisense

probe. For a Smad2 sense probe, pBluescript II SK was cut with EcoRI and transcribed with T3

polymerase. A 602 bp fragment of murine Smad7 cDNA subcloned into pCR2.1 was digested

with Hind III and transcribed with T7 polymerase for an antisense probe. The RNA probes were

labeled with digoxigenin labeling kit (Boehringer Mannheim). Serial sectioned tissue preparation

and subsequent hybridization were performed according to the method of Zhao et al. (25).

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Immunohistochemistry

Sectioned immunohistochemistry was accomplished by following standard procedure (22). In

particular, adjacent sections through the same tooth organ were mounted onto the same slide so

that some of these sections could be stained with either anti-Smad2, or anti-Smad3 (Santa Cruz

Biotech., CA), or PS2 antibody (Dr. C.-H. Heldin) and the others stained with anti-Smad7 (Dr. S.

Souchelnytskyi) to examine the co-localization of different Smads. Positive staining was

indicated by orange-red coloration. The slides were counter-stained with hematoxylin.

Antisense experiments

Smad2 antisense oligodeoxynucleotides (ODN) 5’- GCACGATGGACGACAT -3’ and Smad7

ODN 5’- GTTTGGTCCTGAACAT –3’ were synthesized by Sigma Genosys (The Woodlands,

TX). Because of the high homology between Smad2 and Smad3, only one mismatch at the

underlined nucleotide “C” was found for murine Smad3 cDNA (9, 6). The antisense ODN was

designed to surround the translation initiation site. Two same-length control ODNs (sense and

scrambled) were also synthesized for either Smad2 or Smad7. The first three and the last three

bases in each ODN sequence were modified by phosphorothioation to improve stability against

nuclease degradation in culture. Antisense, sense, or scrambled ODNs were added to E11

mandibular explant cultures in aqueous solution to achieve a final concentration of 30 µM, a

concentration known to be effective in our organ culture system (3, 22). The ODN in fresh

medium was replaced every other day. Each experimental group had 72 mandibular explants.

Morphological, statistical and computer (3-D reconstruction) analysis

In order to evaluate the morphological effect induced by Smad antisense treatment on tooth

formation, the size of tooth organ was measured. Six mandibular explants from each treatment

group (control, Smad2 or Smad7 antisense, sense, or scrambled) were serially sectioned at 5 µm

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intervals and were processed for H and E staining. Each section of a tooth organ was traced

throughout the serial sections of the mandibular explant. The volume of each tooth organ was

determined by adding all the tooth bud area on each serial section and then multiplying by 5 µm

to obtain the total volume of a tooth organ in µm3. Using the Epistat Statistical Package, one-

way, one-level analysis of variance (ANOVA) was applied to test for significant changes in the

size of tooth bud among control, Smad2 or Smad7 antisense ODN, sense ODN and scrambled

ODN treated groups. A difference was considered statistically significant if the p value was less

than 0.05 (p<0.05). To further analyze tooth organ size, three-dimensional reconstructions were

completed as previously described (27).

Western analysis

The total protein concentration in each mandibular sample was determined by comparing to

BSA standards. Seventy-five micrograms total protein from each sample was loaded in each

well on a 12% polyacrylamide gel. Western analysis was done as previously described (22).

Evaluation of DNA synthesis activity during early tooth morphogenesis

DNA synthesis activity was monitored in E11+7 mandibular explants using BrdU (5-bromo-

2’-deoxy-uridine, Sigma) at 100 µM in serumless chemically defined medium for 2 hours at

370C. Mandibular explants were placed in tissue culture dish with either control medium or

medium plus Smad sense or antisense ODN. After two hours culture with BrdU, the mandibular

explants were harvested and fixed for immunostaining (28). BrdU labeled cells and the total

number of cells within the enamel organ epithelium, or within the adjacent dental mesenchyme

of a tooth bud were counted from seven randomly selected sections per mandibular explant.

Three mandibular explants were evaluated from each experimental group.

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Apoptosis assay during early tooth morphogenesis

Apoptotic cells were localized by detecting DNA fragmentation. TUNEL analysis was

performed on paraffin sections using the In Situ Cell Death Detection (Fluorescein) kit

(Boehringer-Mannheim) by following the manufacturer’s protocol.

Preparation of TGF-ββββ and activin beads

Affi-gel blue beads (Bio-Rad), diameter 50-80 µm, were used. The beads were washed in PBS

and then incubated for one hour at room temperature in 10 µg/ml TGF-β2 or activinβA (R&D).

Control beads were incubated in 0.1% BSA.

Genotype analysis

Genotypes of Smad2 and Smad3 mutant mouse embryos were determined by PCR as

previously described (29, 30).

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RESULTS

Spatial and temporal distribution of Smad2, 3 and Smad7 during early tooth

morphogenesis

To investigate the function of each Smad during early tooth morphogenesis, we first examined

the spatial and temporal distribution of Smad2, 3 and Smad7 by both in situ hybridization and

immunohistochemistry. In vitro analysis using E11 mandibular explants cultured in serumless,

chemically-defined medium indicated that Smad2, 3 and Smad7 were closely associated with

tooth morphogenesis from dental lamina stage to late bud stage. In particular, at E11+2, Smad2

was localized within enamel organ epithelium (Fig. 1A), while Smad7 was not detectable (Fig.

1B). At E11+4, Smad3 was localized mainly to the enamel organ epithelium with little staining

in the adjacent mesenchyme (Fig. 1C). Smad7 was expressed within enamel organ epithelium

(Fig. 1D). By the time the mandibular organ culture was terminated (E11+9), tooth development

reached bud stage. Both Smad2 and Smad7 were localized within enamel organ epithelium and

adjacent mesenchyme (Figs. 1E and F). More importantly, phosphorylated Smad2 was localized

within the nuclei of enamel organ epithelial cells, indicating that activated Smad2 had

translocated into the nucleus (Fig. 1E, insert). In tandem, the expression of Smad2 and Smad7

mRNA was also examined. As shown in Figs. 1G and H, the mRNA of Smad2 and Smad7 was

localized to the late bud stage enamel organ epithelial cells at E11+9, a pattern identical to the

immunolocalization of these two Smads (Figs. 1E and F). Furthermore, examination of Smad2, 3

and 7 expression patterns in vivo revealed identical information during these early comparable

stages of tooth development (data not shown). In addition, we also examined the expression

pattern of Smad2 and Smad7 during cap and bell stages of tooth development in vivo (data not

shown). Both of these Smads were mainly expressed in inner enamel epithelium and its adjacent

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dental mesenchyme, indicating that TGF-β signaling plays a critical role in regulating continued

epithelial-mesenchymal interaction throughout tooth development.

Inhibition of endogenous Smad2 and Smad7 gene expression using antisense

oligodeoxynucleotide (ODN) during mandibular morphogenesis in vitro

To investigate the functional role of endogenous Smad2 and 7 during early tooth development,

we utilized the antisense inhibition strategy during mandibular morphogenesis in vitro. Both

competitive PCR and Western analysis were used to measure the amount of Smad expression in

mandibular explants after antisense ODN treatment. All PCR products were sequenced or

digested with restriction enzymes for final verification.

E11 embryonic mandibular explants treated with Smad2 antisense ODN significantly

decreased the amount of endogenous Smad2 and moderately reduced the level of Smad3 mRNA

(Fig. 2a), while Smad2 sense and scrambled control ODNs treated explants yielded amounts of

Samd2 and Smad3 mRNA comparable to the media control (Fig. 2a). Addition of exogenous

TGF-β2 (2 ng/ml) did not reverse the reduction of Smad2 or Smad3 mRNA caused by Smad2

antisense ODN treatment. When exogenous TGF-β2 was added along with sense or scrambled

control ODNs the mRNA level of both Smad2 and Smad3 was not affected.

As illustrated by competitive PCR, mandibular explants cultured with scrambled control ODNs

to Smad7 yielded a comparable amount of Smad7 mRNA to the media control, whereas

mandibles treated with antisense ODN to Smad7 resulted in a reduced level of endogenous

Smad7 mRNA (Fig. 2b). Exogenous TGF-β1 or β2 (2 ng/ml) stimulated endogenous Smad7

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expression in cultured mandibular explants in the presence of either media or scrambled Smad7

ODN control, confirming the responsiveness of Smad7 gene expression to TGF-β stimuli (Fig.

2b). However, when mandibular explants were treated with both Smad7 antisense ODN and

exogenous TGF-β the basal level of Smad7 mRNA was significantly reduced, indicating the

effectiveness of Smad7 antisense oligos. Furthermore, to ensure that Smad7 antisense ODN

specifically inhibited endogenous Smad7 mRNA expression but not other Smad gene expression,

endogenous Smad3 mRNA was examined in the cultured mandibular explants treated with

Smad7 ODN and was not affected (Fig. 2b), indicating the specificity of Smad7 antisense ODN

in mandibular organ culture.

To evaluate the effect of antisense oligos on Smad protein expression, total protein was

extracted from cultured mandibular explants. Smad2 antisense ODN resulted in a significant

reduction of Smad2 and moderate reduction of Smad3, while mandibular explants treated with

sense ODN showed Smad2 and Smad3 expression level comparable to the media control (Fig.

2c). Furthermore, Smad2 antisense ODN also significantly reduced the level of phosphorylated

Smad2 (PS2), indicating a successful interference of TGF-β signaling (Fig. 2c). The same

membrane was striped and incubated with β-actin antibody and showed equal loading of total

protein extract. In another experiment where mandibular explants were treated with Smad7

antisense ODN the expression of Smad7 was significantly reduced, whereas Smad7 sense ODN

treated samples yielded a comparable expression of Smad7 to the control (Fig. 2c). Collectively,

Smad2 and Smad7 antisense ODNs at 30 µM effectively attenuated their gene expression.

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Alteration of endogenous Smad2 and Smad7 gene expression affected TGF-ββββ-mediated

regulation of early tooth development

Attenuation of Smad2 induced a threefold increase (p<0.05) in tooth size as well as an

advanced stage of tooth development (Fig. 3B) compared to non-treated (Fig. 3A) and sense

controls. The addition of exogenous TGF-β1 or β2 to the antisense treated explants did not

restore the tooth size and stage back to a state comparable to the non-treated and sense controls

(see Fig. 7). Our previous experiments, however, demonstrated the effectiveness of addition of

exogenous TGF-β2 to rescue the phenotype caused by TGF-β2 antisense treatment (3). The

extent of advancement of tooth formation with the addition of Smad2 antisense ODN was further

demonstrated with three-dimensional reconstruction of serially sectioned mandibular explants.

As shown in Fig. 4C, the advancement of tooth formation with Smad2 antisense ODN treatment

was not limited to a few sections, but rather it was the entire tooth organ (with an average size of

5.9 X 105 µm3) in comparison to the control (Fig. 4A, with an average tooth organ of 1.7 X 105

µm3) or sense ODN treated (Fig. 4B). Collectively, these results support the conclusion that

attenuation of Smad2 expression releases the negative regulation on tooth growth by endogenous

TGF-β signaling system.

Smad7 antisense treated explants resulted in a significant reduction in the size of tooth organ

(Fig. 3C) when compared to the control (p<0.05), while explants treated with sense ODN did not

show any change in the size and stage of tooth development. Three-dimensional reconstruction

of serially-sectioned mandibular explants treated with Smad7 antisense ODN demonstrated a

more than two-fold reduction in the size of tooth organs (with an average size of 0.5 X 105 µm3)

within the mandible (Fig. 4D). In tandem, our previous dose-response experiments demonstrated

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that addition of exogenous TGF-β2 into mandibular explants reduced the size of tooth organ

approximately threefold (3). Collectively, attenuation of Smad7 gene expression resulted in

elevated TGF-β signaling and a concomitant potentiation of TGF-β-mediated inhibition of early

embryonic tooth development.

Differential activities of Smads help to achieve TGF-ββββ mediated enamel organ epithelial cell

proliferation and death during tooth development.

The biological importance of TGF-β signaling Smads during early tooth development was

evaluated for its impact on enamel organ epithelial cell proliferation. TGF-β-induced growth

arrest occurs late in the G1 phase and is accompanied by a reduction in the cell proliferation

activity in mammalian cells. In this study, DNA synthesis was monitored in E11 mandibular

explants cultured for 7 days in chemically-defined medium. Immunodetection of 5’-bromo-2’-

deoxyuridine (BrdU) labeling was used to identify epithelial or mesenchymal cells engaged in

DNA synthesis. As shown in Fig. 5, E11+7 mandibular explants treated with Smad2 antisense

ODN resulted in advancement of tooth formation with increased BrdU labeled enamel organ

epithelial cells (Fig. 5C) in comparison with the control group (Fig. 5A). Quantitation of the

percentage of BrdU labeled cells showed that attenuation of Smad2 significantly (p<0.05)

increased proliferation within enamel organ epithelium (by more that 50%, see Fig. 5D and Table

1). Meanwhile, there was no significant change of DNA synthesis activity in mesenchymal cells

adjacent to enamel organ epithelium (data not shown).

Smad7 antisense ODN treatment reduced the size of tooth organ without affecting the

proliferation rate of enamel organ epithelial cells (Fig. 5B and Table 1). When we examined the

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apoptotic activity, however, there was a significant increase in cell death in the center part of

enamel organ epithelium in mandibular explants treated with Smad7 antisense ODN (Fig. 6D)

when compared to the non-treated control (Fig. 6B). Smad2 antisense ODN treated mandibular

explants showed advancement of tooth development (Fig. 6E). But there was no detectable

apoptotic activity within the tooth organ (Fig. 6F). Moreover, neither Smad2 nor Smad7 sense

ODN treatment affected the enamel organ epithelial cell proliferation or death rate when

compared to the control (data not shown).

To further explore the association between TGF-β signaling and apoptosis, beads bearing TGF-

β2 (10 µg/ml) were implanted into E11+7 mandibular explants, which were harvested 24 hrs later

for TUNEL assay. There was no detectable apoptotic activity in mandibular explants treated with

BSA bearing beads (Fig. 6H) while there was significant cell death in both epithelium and

mesenchyme adjacent to TGF-β bearing beads (Fig. 6J), indicating that TGF-β induces apoptosis

in mandibular explants. Because Smad2 and 3 are also implicated in activin signaling pathway

we performed experiments to examine the association of activin signaling and apoptosis. Beads

bearing activin (10 µg/ml) were implanted into E11+7 mandibular explants that were harvested

24 hrs later for TUNEL assay. There was no apoptotic activity in dental epithelium while there

was strong apoptosis in the mesenchyme (Fig. 6L). Since there is no activin expression in the

early dental epithelium (31), and the different Smads antisense ODN treatment mainly affected

TGF-β mediated enamel organ epithelial cell proliferation and apoptosis it appears that alteration

of Smad 2, 3, and 7 expression level mainly affected TGF-β mediated signaling during early

tooth development. Recently, mesenchymal activin signaling has been shown to regulate TNF

receptor expression and may interact with Wnt signaling pathway during tooth development (32).

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Clearly, further studies will provide important information on the relationship among TGF-β,

activin and BMP signaling during early tooth development.

In order to test that Smad2 and Smad7 antisense ODN treatment might differentially interfere

with TGF-β-mediated enamel organ epithelial cell proliferation and apoptosis, respectively, we

added exogenous TGF-β along with Smad2 or 7 antisense ODN in cultured mandibular explants.

Tooth development advanced into cap stage with increased enamel organ epithelial cell

proliferative activity in Smad2 antisense ODN plus TGF-β2 (2 ng/ml) treated E11+7 mandibular

explants (Fig. 7B) when compared to the bud stage tooth organ in the control group (Fig. 7A).

Addition of exogenous TGF-β along with Smad2 antisense ODN did not induce apoptosis in the

tooth organ (Fig. 7E) when compared to the control. Smad7 antisense ODN treatment retarded

the tooth development due to increased apoptotic activity within enamel organ epithelium.

Addition of exogenous TGF-β2 (2 ng/ml) along with Smad7 antisense ODN did not affect the

proliferation (Fig. 7C) but resulted in increased apoptotic activity (Fig. 7F) of enamel organ

epithelium. Thus, attenuation of Smad2 interfered with TGF-β-mediated enamel organ epithelial

cells’ proliferative activity while attenuation of Smad7 affected TGF-β-mediated apoptotic

activity.

Heterozygous loss of Smad2 or Smad2 and 3 are sufficient to alter TGF-ββββ mediated tooth

development.

Targeted mutations of both Smad2 and Smad3 have been previously described (33, 30).

Smad2 homozygous mutant embryos fail to form an organized egg cylinder and lack mesoderm.

Interestingly, some of the Smad2 heterozygous embryos have severe gastrulation defects and lack

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mandibles or eyes. Smad3 homozygous mutant mice have impaired mucosal immunity and

diminished T cell responsiveness to TGF-β, but without any obvious developmental defects. E11

mandibular explants isolated from Smad2+/-, Smad3+/-, or Smad2+/- and Smad3+/- (Smad2+/-

/Smad3+/- compound heterozygous mutant) mouse embryos were cultured in serumless,

chemically-defined medium for nine days. All mandibular explants from Smad3+/- (n=51) mouse

embryos formed bud stage tooth organ (Fig. 8B), as seen in the wild-type littermate controls (Fig.

8A). In cultured mandibular explants obtained from Smad2+/- (n=44) or Smad2+/-/Smad3+/-

(n=18) mouse embryos, however, about 30 % to 40% of the tooth development advanced into late

cap stage (Figs. 8C & D). Hence, functional haploinsufficiency of Smad2, but not Smad3,

resulted in alteration of tooth development, indicating that the dosage of Smad2 is critical for

TGF-β signaling during tooth morphogenesis.

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DISCUSSION

Members of the Smad family of signal transduction molecules are components of a critical

intracellular pathway that transmits TGF-β signals from the cell surface to the nucleus. Targeted

disruptions of Smad genes have revealed important biological functions of these intracellular

signaling molecules. Many of the Smad null mutations, however, resulted in early embryonic

lethality, indicating the biological importance of the Smad protein in TGF-β signaling although

preventing the functional analysis of Smad molecule during organogenesis (32, 29, 30, 34, 35).

The present study seeks to understand the biological function of both receptor-regulated and

inhibitory Smads in regulating TGF-β-mediated early tooth development. Previous studies

indicated that TGF-β and its cognate receptors are important regulators during early tooth

development (36, 37, 3, 22). Specifically, endogenous TGF-β signals through its cognate

receptors and exerts negative control on the proliferation of enamel organ epithelium to regulate

the overall tooth growth during early tooth development (22). The mechanism of negative

regulation, however, on the proliferation of enamel organ epithelium by TGF-β is not

understood. Despite the various craniofacial malformations associated with TGF-β2 or 3 null

mutant, there is no tooth abnormality reported in any of the TGF-β knockout mice, indicating

that there are possible functional overlaps among various isoforms of TGF-β (38-41). This

theory is further supported by TGF-β type II receptor null mutation, which is early embryonic

lethal and demonstrates the severe consequence of blocking all TGF-β isoforms signaling (42).

In tandem, activin is an early essential dental mesenchyme signal required for tooth development

(31). Recent study has demonstrated that the expression of Smad2 and Smad3 are present in

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epithelially derived ameloblasts and cranial neural crest derived odontoblasts, indicating that

TGF-β/activin are critical regulators during tooth development (23, 43, 56). Our study showed

that both positive and negative TGF-β signaling regulatory Smads were expressed during early

tooth development in a similar spatial and temporal pattern (Fig. 1). Using the PS2 antibody, we

demonstrated that phosphorylated Smad2 was present in the nuclei of enamel organ epithelial

cells, indicating the active role of Smad2 in regulating tooth development. In a recent study,

using the same PS2 antibody, overexpression of Smad7 transgene was able to block Smad2

phosphorylation induced by bleomycin in mouse lungs, validating the specificity of this antibody

in recognizing the activated Smad2 in mouse tissue (44). More importantly, the spatial and

temporal distribution of Smad2, 3 and 7 matched precisely with the distribution of TGF-β ligand

and its cognate receptors during early tooth development, suggesting the possible regulatory role

of these intracellular signaling molecules (36, 37, 3, 22). When we compared the expression

patterns of Smads (2, 3, and 7) and activin (31), however, it appeared that these Smads were

more closely associated with TGF-β mediated early enamel organ epithelium development.

The expression of Smad7 was rapidly induced by the addition of exogenous TGF-β, indicating

the negative feedback on TGF-β signaling during mandibular morphogenesis in vitro. This

negative regulation on TGF-β signaling was likely achieved by the competitive binding between

Smad2, 3 and Smad7 to the TGF-β type I receptor (17, 45). Smad7 can bind onto the GS domain

on type I receptor and prevent the phosphorylation of Smad2 upon activation by TGF-β ligand.

In our recent study using adenovirus to overexpress Smad7 during mandibular morphogenesis in

vitro we found a significant reduction of phosphorylated Smad2 (unpublished data), indicating

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that Smad7 might prevent the phosphorylation of Smad2 by TGF-β type I receptor and function

as a negative regulator for TGF-β signaling.

To investigate the specific regulatory function of Smad2 and Smad7 during early tooth

development we designed antisense oligodeoxynucleotides to attenuate the Smad gene

expression on both transcriptional and translational levels during mandibular morphogenesis in

vitro. The antisense ODNs were strategically designed near the translation initiation codon, the

sequence known empirically to be most effective in inhibiting translation of target mRNA (46).

Previous studies have shown that short ODNs diffuse into mammalian cells, hybridize to target

transcripts, and effectively degrade mRNA without any noticeable cytotoxicity (47, 48, 3, 22).

Recent studies using similar approach have demonstrated that Smads were critical regulators of

TGF-β signaling during lung branching morphogenesis in vitro (26, 49).

In this study, Smad2 antisense ODN was able to significantly attenuate Smad2 expression and

reduce the phosphorylation of Smad2. Consequently, tooth development advanced from the bud

to the cap stage during mandibular morphogenesis in vitro. This observation was confirmed by

using mandibular explants from either Swiss-Webster or C57BL/6 mice (on which background

the Smad2 and Smad3 mutants are maintained). Cell proliferation analysis indicated Smad2

antisense ODN treatment significantly increased the proliferation of enamel organ epithelial cells

and resulted in enlargement of tooth organ while apoptosis assay showed no detectable cell death

in the tooth bud. Previous study had shown that both Smad2 and Smad3 could strongly induce

apoptosis in lung epithelial cells (50). Hence, attenuation of Smad2 reduced the inhibition on

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proliferation of enamel epithelial cells mediated by TGF-β and resulted in the advancement

during early tooth development.

TGF-β signaling regulates cell proliferation as well as apoptosis. Recently, Smad7 has been

shown to play an important role in regulating TGF-β-mediated apoptosis (4, 51). Here, we

demonstrated that attenuation of Smad7 resulted in a significant increase of apoptosis within

enamel organ epithelium. Similarly, beads bearing TGF-β implanted within the mandibular

explant also resulted in elevated apoptotic activity within enamel organ epithelium, indicating

that increased TGF-β signaling (either by attenuation of TGF-β signaling inhibitory Smad or by

addition of exogenous TGF-β) may induce TGF-β-mediated apoptosis. Interestingly, elevated

Smad7 expression (e.g. induced by IFNγ) promotes Smad7-Smurf2 complex formation and,

consequently, increases TGF-β receptor turnover (57). It is, therefore, tempting to suggest that

attenuation of Smad7 expression may interfere with the Smad7-Smurf2 mediated TGF-β receptor

turnover and potentiate TGF-β induced apoptotic activity. Importantly, this study showed, for

the first time, that an alteration in the expression of receptor activated Smad or inhibitory Smad

may determine the same type of cells either continues with proliferation or goes through

apoptosis.

Due to the high homology between Smad2 and Smad3 we were not able to design a specific

ODN which would only reduce the expression of either Smad2 or Smad3, but not together. Our

study showed, however, that Smad2 antisense ODN significantly reduced Smad2 expression but

only resulted in marginal reduction of Smad3. Interestingly, using Smad2+/- and Smad3+/-

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heterozygous as well as Smad2+/-/Smad3+/- compound heterozygous mutant mouse embryos, we

demonstrated that Smad2, but not Smad3, played an important regulatory role for TGF-β

signaling during early tooth development. More importantly, our results indicate that the dosage

of Smad2 gene expression is critical for TGF-β signaling. In some of the cultured mandibular

explants taken from Smad2+/- and Smad2+/-/Smad3+/- heterozygous mutant mouse embryos, it is

probable that the signals transduced by Smad2 is below the threshold and the negative regulation

on enamel organ epithelial cells is interrupted which resulted in the advancement of tooth

development in vitro.

We did not see any alteration of tooth development in cultured mandibular explants taken from

Smad3 heterozygous mutant mouse embryos. Smad3 null mutant mice develop normally. Adult

Smad3-/- mice have impaired mucosal immunity and diminished T cell responsiveness to TGF-β

as well as form metastatic colorectal cancer (52, 30). Because of the high homology between

Smad3 and Smad2 (> 95%) it has been speculated that the shorter form of Smad2 (alternatively

spliced Smad2 without exon 3) may function as Smad3 in transducing TGF-β signaling, thus, can

compensate for the Smad3 null mutation (53, and Chuxia Deng, personal communication).

Recently, Smad3 has been shown to play a more important role in TGF-β mediated pathogenetic

events and be less involved during embryonic development (54). Further studies are needed to

investigate the possible functional uniqueness of Smad3 in regulating TGF-β signaling.

The TGF-β signaling pathway is regulated in both positive and negative fashions, and is

tightly controlled temporally and spatially through multiple mechanisms at the extracellular, cell

membrane, cytoplasmic and nuclear levels (4, 55, 45). At present, positive regulation of TGF-β

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signaling is carried out by the phosphorylation of Smad2 and Smad3 and is a critical control for

amplification of signaling. Negative regulation by Smad7 also plays an important role in the

restriction and termination of signaling. This negative feedback could be a critical limiting factor

on the range of TGF-β signaling and form a gradient to precisely regulate TGF-β ligand activity

during organogenesis. This study, along with our previous study on TGF-β type II receptors,

clearly demonstrates the multiple levels of TGF-β signaling regulation and begins to address how

the size of tooth organ is controlled during early craniofacial development (3, 22).

We conclude that endogenous TGF-β peptide signaling through not only TGF-β receptors, but

also Smads, exerts negative control on the proliferation of enamel organ epithelium. The

negative regulation is mediated by intracellular Smads in a dose-dependent manner. Functional

haploinsufficiency of Smad2 causes alteration in early tooth development. Smad7, however, is

critical for regulating TGF-β induced apoptosis during mandibular morphogenesis in vitro. It is

possible that Smads, together with other transcriptional activators/repressors, orchestrate TGF-β-

mediated gene regulation during embryonic organogenesis. We hypothesize that endogenous

TGF-β signaling through the Smads may serve to negatively modulate and therefore balance the

positive functions of signaling by other peptide growth factor pathways, such as FGF, BMP and

PDGF, which exert inductive and/or permissive influences on tooth morphogenesis.

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ACKNOWLEDGEMENT

We thank Dr. Carl Heldin and Dr. Serhiy Souchelnytskyi for providing the Smad2, 3 and 7

antibodies. Ms. Cindy Woo’s technical assistance is highly appreciated. We would also like to

thank the two referees for their invaluable insights and suggestions during the review process.

This study was supported by grants from the National Institute of Dental and Craniofacial

Research, NIH (DE12711 and DE12941 to Y. C.).

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FIGURE LEGENDS

Figure 1. Expression of Smad2, Smad3 and Smad7 during early stages of tooth morphogenesis in

vitro. (A) At E11+2, Smad2 was present in dental lamina (arrow). tb = tooth bud. (B) The same

tooth organ was sectioned and stained with anti-Smad7 antibody. There was no detectable Smad7

in the dental lamina. After 4 days in culture, Smad3 (C) and Smad7 (D) were localized mainly in

the enamel organ epithelium (arrow). By the end of 9 days, (E) Smad2 (PS2) was specifically

associated with bud stage enamel organ epithelium (tb), but not with the rest of oral epithelium

(*). Because PS2 antibody could recognize phosphorylated Smad2 and the positive signal of PS2

was mainly found within the nuclei of enamel organ epithelium (arrow and insert) it was

conceivable to assume that TGF-β signaling played an active role in regulating the development

of enamel organ epithelium. (F) The same serially-sectioned tooth organ showed the expression

of Smad7 within enamel organ epithelium, with positive staining mainly in the cytoplasm (arrow).

Some of the serial sections from the same tooth organ were used for in situ hybridization. At

E11+9, (G) Smad2 mRNA was localized in the enamel organ epithelium (arrow), but not in the

oral epithelium (*) supporting the immunolocalization data shown in E. (H) Smad7 mRNA was

present in the bud stage enamel organ epithelium (arrow). Scale bar = 50 µm.

Figure 2. Inhibition of Smad2, Smad3 and Smad7 gene expression by antisense

oligodeoxynucleotides (ODN). E11 mandibular explants were treated with plain media as control

(MC), scrambled (SR) ODN at 30 µM or antisense ODN at 30 µM to Smad2 mRNA. (a) Smad2

and Smad3 mRNA was greatly reduced in Smad2 antisense ODN treated groups compared to

scrambled ODN treated or the control groups. Addition of exogenous TGF-β1 at 2 ng/ml (+) did

not reverse the reduction of Smad2 and Smad3 mRNA caused by Smad2 antisense ODN

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treatment. The addition of exogenous TGF-β1 along with scrambled ODN did not affect the

expression of endogenous Smad2 or Smad3. To verify that there was no variation due to the

efficiency of RNA extraction and reverse transcription, β-actin mRNA was quantified in the same

samples and showed no difference. (b) Smad7 antisense oligodeoxynucleotides (ODN)

effectively inhibits its mRNA expression in cultured mandibular explants. Exogenous TGF-β1 or

β2 induced Smad7 mRNA expression in cultured mandibular explants in the presence of either

media control (MC) or Smad7 scrambled (SR) control ODN. However, Smad7 antisense (AS)

ODN not only inhibited the basal level of Smad7 mRNA expression in the absence of exogenous

TGF-β1, but also attenuated TGF-β1-mediated Smad7 up-regulation in mandibular explants in

vitro. Smad7 antisense ODN treatment specifically attenuated its own mRNA expression and did

not adversely affect the expression of Smad3 mRNA. (c) Smad antisense ODN also decreased its

protein level in E11+9 cultured mandibular explants. Antisense (AS) ODN to Smad2 reduced

Smad2, phosphorylated Smad2 (PS2), and Smad3 protein levels comparing to sense (SE) ODN

treated or media control (C). The level of β-actin indicated that equal amount of total protein was

loaded in each lane for the Western analysis. Smad7 antisense ODN also reduced its protein level

comparing to either control (C) or sense ODN treatment.

Figure 3. Attenuation of endogenous Smad2, 3 and 7 gene expression altered TGF-β-mediated

regulation of tooth morphogenesis. (A) At E11+9, cultured mandibular explants showed bud

stage tooth formation. (B) Smad2 antisense ODN treated mandibular explants showed an

advancement of tooth formation to the cap stage and increased size of the tooth organ at E11+9.

(C) Smad7 antisense ODN treated mandibular explants resulted in retardation of tooth

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development, with a two-fold reduction in the size of enamel organ epithelium. Scale bar = 50

µm.

Figure 4. Three-dimensional reconstructions from serially sectioned mandibular explants were

analyzed to define the overall size of tooth organ in E11+9 mandibular explants. (A) Control. (B)

E11+9 mandibular explants treated with Smad2 sense ODN. The size of both incisor and molar

tooth organs were comparable to the ones in the control group. (C) Smad2 antisense ODN

treated mandibular explants advanced the size and stage of both incisor and molar tooth

formation by more than two-fold. (D) Smad7 antisense ODN treated mandibular explants showed

retardation of tooth formation (incisors and molars). Tooth organs were indicated in red.

Meckel’s cartilage was reconstructed in blue. The outlines of serially-sectioned mandibular

explant was shown in green. Sectioned mandibular explants were reconstructed with the outlines

being transparent and presented with increased intervals of the outlines to show the tooth and

Meckel’s cartilage position and size.

Figure 5. Attenuation of endogenous Smad2 increased the proliferation of enamel organ

epithelial cells. (A) At E11+7, the tooth organ was at bud stage with BrdU labeled cells

indicating DNA synthesizing activity. Note the center part of enamel organ was filled with cells

(arrow). (B) Attenuation of Smad7 did not affect the cell proliferation rate within enamel organ

epithelium that mainly occurred at the peripheral of the enamel organ epithelium (double arrow).

Note the void within the center part of enamel organ epithelium (signal arrow), which might

resulted from increased apoptosis in this area (see Fig. 6). (C) Attenuation of Smad2

significantly (p < 0.05) increased the proliferation of enamel organ epithelial cells. (D) As a

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result of cell count, only 19% of total counted cells within enamel organ epithelium were labeled

with BrdU in the control group. Smad2 antisense treated mandibles showed a significant

increase of BrdU labeled cells (33%) within enamel organ epithelium, while Smad7 antisense

treatment did not affect the proliferation (21%) of enamel organ epithelial cells.

Figure 6. TGF-β induced apoptosis during mandibular morphogenesis in vitro. (A) E11+7

cultured mandibular explants showed bud stage tooth development (t). (B) TUNEL assay

revealed no apoptotic activity within enamel organ epithelium (t). A very limited apoptotic

activity was detected at the surface oral epithelium (*). (C) E11+7 mandibular explants treated

with Smad7 antisense ODN showed inhibition of tooth development. (D) TUNEL assay

revealed significantly increased apoptotic activity within the center part of enamel organ

epithelium (single arrow), while the peripheral region had no detectable cell death (double arrow)

in Smad7 antisense ODN treated explant. (E) E11+7 mandibular explants treated with Smad2

antisense ODN showed advancement of tooth development (t). (F) TUNEL assay revealed no

apoptotic activity within the enamel organ epithelium (outlined by the dotted line) in Smad2

antisense ODN treated explant. (G) Bead bearing 0.1% BSA implanted within E11+7

mandibular explant for 24 hours. (H) TUNEL assay revealed no detectable apoptotic activity

adjacent to the bead. The dotted line indicates basement membrane of ectoderm. (I) Bead

bearing TGF-β2 (10 µg/ml) implanted within E11+7 mandibular explant for 24 hours. (J)

TUNEL assay revealed significant apoptotic activity adjacent to TGF-β bearing bead, especially

in oral ectoderm (above the dotted line). (K) Bead bearing activinβA (10 µg/ml) implanted

within E11+7 mandibular explant for 24 hours. (L) TUNEL assay revealed significant

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mesenchymal apoptotic activity adjacent to activin bearing bead. There is no apoptotic activity

detected in oral ectoderm.

Figure 7. Smad2 and Smad7 antisense ODN treatment differentially interfered with TGF-β-

mediated enamel organ epithelial cell proliferation and apoptosis, respectively. (A) At E11+7,

cultured mandibular explant showed proper enamel organ epithelial cell proliferation in the

control tooth organ (tb). (B) Smad2 antisense ODN plus exogenous TGF-β2 (2 ng/ml) treated

mandibular explant showed advanced tooth development into cap stage with significantly

increased proliferative activity within the enamel organ epithelium (*). (C) Smad7 antisense

ODN plus exogenous TGF-β2 (2 ng/ml) treatment retarded tooth development without affecting

the proliferation of enamel organ epithelial cells (*). (D) At E11+7, bud stage tooth organ (tb)

did not show any apoptotic activity in the control sample. (E) Smad2 antisense ODN plus

exogenous TGF-β2 (2 ng/ml) treated mandibular explant showed advanced tooth development

into cap stage without any detectable apoptotic activity within the enamel organ epithelium. (F)

Smad7 antisense ODN plus exogenous TGF-β2 (2 ng/ml) treatment retarded tooth development

with increased apoptotic activity in enamel organ epithelial cells (arrow).

Figure 8. Heterozygous loss of Smad2 and Smad3 were sufficient to alter TGF-β signaling

mediated tooth development. (A) At E11+9, cultured mandibular explants of wild type mice

showed bud stage tooth formation (tb) (B) Cultured mandibular explants of Smad3+/-

heterozygous mutant mice showed a bud stage tooth organ at E11+9, comparable to the wild type

control. (C) Mandibular explants of Smad2+/- heterozygous mutant mice showed an

advancement of tooth formation to the cap stage and increased size of the tooth organ at E11+9.

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(D) Smad2+/-/Smad3+/- double heterozygous mutant mice also showed an advancement of tooth

formation to the cap stage and increased size of the tooth organ at E11+9.

Table 1. BrdU labeled cells and total number of cells counted (in parentheses) within enamel

organ epithelium (b). Three E11+7 cultured mandibular explants were randomly chosen from

each group. All sections contained tooth organ. Two adjacent sections were chosen with at least

40 µm distance in between to avoid the same cells being counted twice. Mandibular explants

treated with Smad2 antisense ODN substantially (p<0.05) increased the percentage of BrdU-

labeled cells within enamel organ epithelium. (a) BK 1, 2, and 3 = randomly selected E11+7

cultured mandibular explants 1, 2, and 3.

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Deng and Yang ChaiYoshihiro Ito, Jingsong Zhao, Ali Mogharei, Charles F. Shuler, Michael Weinstein, Chuxia

during tooth developmentAntagonistic effects of Smad2 versus Smad7 are sensitive to their expression level

published online September 13, 2001J. Biol. Chem.

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antagonistic effects of smad2 versus smad7 are sensitive ... · 9/13/2001 · jbc m0-11424 the...

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