Influence of ADAM28 on biological characteristics of humandental follicle cells

Zheng Zhao a,b, Hongchen Liu a,*, Yan Jin c, E. Lingling a

a Institute of Stomatology, General Hospital of Chinese People’s Liberation Army, 28 Fuxing Road, Beijing, 100853, ChinabDepartment of Stomatology, The 401st Hospital of Chinese People’s Liberation Army, Qingdao, 266071, ChinacResearch and Development Centre for Tissue Engineering, Fourth Military Medical University, Xi’an, Shaanxi 710032, China

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5

a r t i c l e i n f o

Article history:

Accepted 17 May 2009

Keywords:

ADAM28

HDFCs

Proliferation

Differentiation

Apoptosis

a b s t r a c t

Objectives: The aim of this study was to investigate the effects of a disintegrin and metallo-

proteinase 28 (ADAM28) on the biological characteristics of human dental follicle cells

(HDFCs) and possible action mechanism.

Methods: Eukaryotic expression plasmid containing ADAM28 coding region and ADAM28

antisense oligodeoxynucleotides (AS-ODN) with FITC labelling were constructed and

synthesised by gene clone and recombination. Then we respectively transfected them into

HDFCs by Lipofectamine 2000 system and detected their effects on proliferation, differ-

entiation and apoptosis of HDFCs by MTT assay, cell cycle detection, ALP activity and

Annexin V-FITC/PI analysis. Finally we observed the effects of ADAM28 AS-ODN on HDFCs

expressing extracellular matrix (ECM) proteins by immunocytochemical staining.

Results: ADAM28 eukaryotic plasmid was constructed and identified successfully, and could

be correctly translated and expressed in HDFCs, furthermore overexpression of ADAM28

promoted the HDFCs proliferation and inhibited specific differentiation of HDFCs, while

inhibition of ADAM28 exerted the opposite effects and induced apoptosis. Moreover

ADAM28 could significantly inhibit the secretion of OPN and type III collagen of HDFCs.

Conclusions: ADAM28 might actively participate in the network regulation which associates

HDFCs proliferation, differentiation, apoptosis with matrix mineralisation during tooth

development by interacting with multiple signal molecules.

Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved.

avai lab le at www.sc iencedi rect .com

journal homepage: www.intl.elsevierhealth.com/journals/arob

1. Introduction

The dental follicle (DF) is a loose ectomesenchymally derived,

connective fibrous tissue sac surrounding the enamel organ

and the dental papilla of the developing tooth germ prior to

eruption. The DF cells are generally believed to contain

precursor cells for cementoblasts, osteoblasts and periodontal

ligament cells and further have the capability to differentiate

into periodontium consisting of cementum, alveolar bone and

* Corresponding author. Tel.: +86 010 83345786; fax: +86 010 66936254.E-mail address: zhaozheng6912@sohu.com (H. Liu).

0003–9969/$ – see front matter . Crown Copyright # 2009 Published bdoi:10.1016/j.archoralbio.2009.05.013

periodontal ligament.1 The development of the tooth root and

periodontium is thought to derive from the DF cells through a

mechanism of epithelial–mesenchymal or extracellular

matrix-mesenchymal interaction.2

Tooth eruption is a complex and tightly regulated process

involved in both odontogenic and osteogenic cells, which

requires alveolar bone resorption and formation, and appears

to be regulated by the DF.3,4 Furthermore, the DF cells of a

developing tooth secrete a bone resorbing factor that can

y Elsevier Ltd. All rights reserved.

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5836

enhance and maintain osteoclast formation, which is needed

for a tooth eruption pathway in the alveolar bone. It is thus

clear that DF cells play an essential role in the process of tooth

root development and tooth eruption.5 Previous studies have

indicated that the biological functions of DF cells are

controlled by a network of regulatory molecules including

growth factors, extracellular matrix proteins (ECM), cytokines

and developmental genes.6,7

ADAM28 gene is a newly discovered member of ADAM

family in humans and murine with proteolytic, adhesive

properties and autocatalytic activity,8,9 which screened from

patients with congenital hypoplasia of tooth root (CHTR) by

the authors.10 CHTR is a disease of tooth root physiological

development disorder resulting from ectodermal dysplasia

and the patients usually display tooth mobility, early slough

and atonia masticatoria. According to clinical situation, CHTR

is divided into maldevelopment of root cementum and dentin,

root paramorphia such as short cone anomaly which occurs

mostly in maxillary incisors, and root attachment organ

dysplasia.11,12 So far no effective therapy has been found in the

world, and its concrete pathopoiesis mechanism still remains

unclear. The human ADAM28 gene is located in chromosome

8p21.2 and is mainly expressed in dendrite-like cells,

lymphocytes and macrophages.13 The defining feature of

the ADAM28 is a conserved modular domain organisation

consisting of a N-terminal signal sequence, a prodomain, a

metalloprotease domain (MP) and a disintegrin-like domain, a

cysteine-rich domain, an epidermal growth factor (EGF)-like

domain, a transmembrane domain and a cytoplasmic

domain.14,15 Furthermore, the MP activity is of functional

importance for extracellular matrix modelling and in the

ectodomain processing of molecules.8

ADAM28 is known to have been implicated in several

crucial biological processes, including neurogenesis, muscle

development, protein ectodomain shedding, cell–cell adhe-

sion events and release of membrane-anchored proteins.16

Researches uncovering the overexpression of ADAM28 in

non-small cell lung carcinomas and breast carcinoma

cell implicated its involvement in the interplay of various

cell–cell and cell–matrix interactions, cell proliferation, and

cell motility.17,18 Recent studies displayed that ADAM28

could promote proliferation of human breast cancer cells by

the IGF signalling, and that the IGF system has a funda-

mental role in protecting cells from apoptosis.19 These

evidence suggested that ADAM28 might be a regulatory

factor closely related to organogenesis, cell proliferation and

apoptosis.

Furthermore, our findings have proved that ADAM28 was

expressed at almost each stage of human and murine tooth

germ development with different levels, and it may participate

in this network regulation to link cell proliferation, differ-

entiation with matrix synthesis.10 However, little attention

has hitherto been paid to the effects of ADAM28 on biological

features of HDFCs from molecular level and functional

mechanisms of relatively independent odontogenic capability

of HDFCs.

Therefore, to elucidate the effects of ADAM28 gene on

biological characteristics of HDFCs, we used immunocyto-

chemistry, molecular biology and cell biology techniques to

study the roles of ADAM28 in HDFCs development.

2. Materials and methods

The nucleotide sequence data about human ADAM28 in this

study appear in the GenBank Nucleotide Sequence Databases

under accession no. NM-014265. ADAM28 polyclonal antibody

was prepared in our laboratory and the specificity and titre of

polyclonal antibody were determined undoubtedly.10 The

protocol was approved by the Ethical Committee on Human

and Animal Research of General Hospital of Chinese PLA.

2.1. Cell culture and source identification

The dental follicles were physically isolated from the lower

third molar tooth germs (uneruption) of 12-year-old patients

under stereomicroscope (Olympus, Microsystems, Japan).

The surfaces of the follicles were cleaned, rinsed in

Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL,

Grand Island, USA) containing penicillin G (100 U/mL,

Gibco-BRL, Grand Island, USA) and streptomycin (100 mg/

mL, Gibco-BRL, Grand Island, USA) and minced into pieces.

The follicles were then digested in a solution of dispase I

(Gibco-BRL, Grand Island, USA) and type I collagenase

(0.66 mg/mL; Sigma, St.Louis, MO) for 1 h at 37 8C.20 Single

cell suspensions were generated by filtration through a 70 mm

strainer, washed with DMEM supplemented with 10% (v/v)

fetal bovine serum (FBS, Gibco, USA), and then placed into

culture flasks and cultured in 5% CO2 at 37 8C. Through

repeated differential trypsinisation as reported,21 purified

HDFCs were obtained excluding the contamination from

epithelial cells. When the primary HDFCs arrived 90%

confluence, they were digested in 0.25% trypsin and

performed subculture. The fourth passage HDFCs were used

for the following experiments. The patients gave informed

consent and donation was voluntary.

Source identification: immunocytochemical staining of

cultured HDFCs with monoclonal antibody (mAb) against

vimentin (DAKO, USA) at 1:30 dilution and mAb against

cytokeratin (DAKO, USA) at 1:150 dilution were performed

respectively.

2.2. The construction and identification of ADAM28eukaryotic expression plasmid

In the present study, human ADAM28 was extracted and

cloned from lymphocytes. A PCR product (2,327 bp) corre-

sponding to total length of ADAM28 coding region (48–2375)

was generated using gene-specific primers:

Upstream primer: 50-ATGTTGCAAGGTCTCCTGCCAGT-

CAGTCTC-30 (amino acids 48–77); Downstream primer: 50-

TCATGCTTTTGGATTTGAGTCCTTAGGTGTAGACA-30 (amino

acids 2375–2341). PCR product was separated by 1% agarose

gel electrophoresis, visualised with ethidium bromide staining

and purified by NucleoTrap Gel Extraction Kit (Clontech, USA).

A 2327 bp fragment was cloned into the corresponding sites

of a pMD18-T vector (Takara, Japan) to construct pMD18-T-

ADAM28. The positive clones containing the target inserts

were sequenced prior to transformation into Escherichia coli

DH5a. DNA homology search was performed on the National

Center for Biotechnology Information (NCBI) server with

BLAST (Basic Local Alignment Search Tool) 2.0 program.

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5 837

Total length of human ADAM28 coding region was excised

from pMD18-T-ADAM28 using EcoR I and Sal I, and ligated into

pcDNA3.1(+) to construct the recombinant plasmid

pcDNA3.1(+)-ADAM28 with EcoR I and Not I digestion. The

eukaryotic expression plasmid was verified by PCR, restriction

endonuclease digestion identification and DNA sequencing.

Meanwhile, glyceraldehyde-3-phosphate dehydrogenase

(GAPDH, 900 bp) was used as a control, specific primers were

as follows: forward: 50-AGCCGCATCTTCTTTTGCGTC-30;

reverse: 50-TCATATTTGGCAGGTTTTTCT-30.

2.3. Transfection of HDFCs with ADAM28 eukaryoticplasmid and expression detections

Experimental cells were divided into three groups according to

pcDNA3.1(+)-ADAM28 group, pcDNA3.1(+) group with

30 mmol/L concentration and untransfected group.

Transient transfection was performed using Lipofecta-

mineTM 2000 system. At 1 day before transfection, the

fourth passage HDFCs were seeded in 24-well culture

plates (Nunc A/S, USA) at a cell density of 5 � 106 with

coverslips put in partial wells, 75 cm2 culture flasks (Gibco,

USA) and 96-well plates (Gibco, USA). Cells were cultivated

in the DMEM/F12 containing 10% FBS but not containing

penicillin G (100 U/mL) and streptomycin (100 mg/mL). At 4 h

before transfection, DMEM/F12 not containing FBS was

added into flasks. 1.0 mg DNA of each well was diluted with

50 mL DMEM/F12 not containing FBS, meanwhile 2 mL

LipofectamineTM 2000 (Invitrogen, USA) of each well

was diluted with 50 mL DMEM/F12 not containing FBS.

After 5 min of incubation at room temperature, diluted

DNA and LipofectamineTM 2000 reagent were combined

and incubated at room temperature for 20 min to form

the DNA-Lipofectamine 2000 complex. Then, 1 mL of

complex was added into 24-well culture plates and flasks

respectively. After 6 h of transfection, routine DMEM/F12

containing 10% FBS was changed into the plates and flasks.

After 48 h, cells on coverslips were washed with PBS, fixed in

4% paraformaldehyde for 2 h and then subjected to

immunofluorescence staining. The procedures were as

follows: After extensive washing with PBS, cells were

incubated with normal goat serum at 37 8C for 30 min and

then exposed to the ADAM28 antibody (1:200 dilution)

overnight at 4 8C. Thereafter, the cells were washed three

times with PBS and incubated with FITC-labelled goat anti-

rabbit IgG (1:50 dilution, Santa Cruz, USA) at 37 8C for

30 min. After being washed with PBS, fluorescence was

observed under microscope (Olympus, Microsystems,

Japan).

The other cells in 24-well plates and 75 cm2 flasks were

collected and used for RT-PCR, Western blot (Western blotting

kit, Chemicon International, Temecula, CA, USA) and cell cycle

detections. GAPDH was used as an endogenous control.

Meanwhile labworks software of UVP gel image analysis

system was used to detect the greyscales of all PCR bands.

Alpha ImagerTM 1220 image analysis system was used to

detect the greyscales of Western blot bands. Relative greyscale

analysis of gene expression was calculated by the Delta CT

method with ADAM28/GAPDH, which was normalised to the

GAPDH controls.

2.4. The design and transfection of ADAM28 AS-ODN

The nucleotides of 20nt specifically targeting human ADAM28

mRNA (GenBank no. NM-014265) were designed and synthe-

sised as ADAM28 AS-ODN (50-GG CAG GAG ACC TTG CAA CAT-

30, 67–48), meanwhile the sequences of 20nt were designed as

ADAM28 S-ODN (50-CAG TCT CCT CCT CTC TGT TG-30, 71–90).

The sequences were subjected to sulphur modification and

FITC fluorescence labelling (Takara, Japan), and confirmed to

have satisfactory specificity by NCBI/BLAST 2.0 database

search. Experimental concentration was 30 mmol/L. Trans-

fection procedures were the same as mentioned before.

Experimental cells were divided into three groups according

to the ADAM28 AS-ODN group, S-ODN group with 30 mmol/L

transfection concentration and untransfected group. Trans-

fection efficiency was observed under fluorescence micro-

scope after transfected 48 h, and transfection

efficiency = fluorocyte number at the same eyeshot/total

cellular score at the same eyeshot under inverted phase

contrast microscope � 100%. The inhibition effect of ADAM28

AS-ODN on HDFCs was determined by immunocytochemical

(LsABC kit, DAKO, USA) staining, RT-PCR and Western blot

assays.

Immunocytochemical staining and image analysis were

performed as described previously.10 The average greyscale

(means � S.D.) and relative greyscale from RT-PCR and

Western blot assays of each group were obtained, and the

results were subjected to statistical analysis.

2.5. Cell proliferation assay

The cell density was kept at 5 � 106 using for the detection of

MTT, cell cycle, apoptosis and ALP activity. Cell proliferation

assay was performed in all five groups of HDFCs using MTT [3-

(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazoliumbro-

mide] method, respectively 20 mL (5 mg/mL) of MTT was

added into each well of 96-well plate for incubation 4 h at

37 8C, after 48 and 72 h of transfection. Then the supernatant

was removed and 150 mL of dimethylsulfoxide (DMSO) was

added following 10 min of oscillation. The optical density (OD)

value was determined with an enzyme-linked immunosor-

bent assay (ELISA) machine at a wavelength of 490 nm and the

assay was repeated for five times. The data were presented as

the means � S.D.

2.6. Cell cycle detection

The cells after 48 h and 72 h of transfection were performed

cell cycle analysis. The cellular DNA content was determined

by flow cytometry, as described previously.22 We collected the

floating and attached cells using trypsin-EDTA and resus-

pended them in DMEM (no FBS). The cells were fixed for 30 min

in an ice-cold 70% ethanol solution containing ribonuclease

(RNase; 2 mg/mL). We washed the cells in PBS, and then

stained them with propidium iodide (PI) for 30 min at room

temperature in the dark. The PI-elicited fluorescence was

measured for individual cells using a FACSCalibur flow

cytometer (Becton Dickinson, Tokyo, Japan) with laser excita-

tion at 488 nm. We analysed a total of 5 � 106 cells for each

sample and determined the percentages of cells in G0/G1, S and

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5838

G2/M phases using standard ModiFit and Cell Quest software

(Becton Dickinson Biosciences).

2.7. Apoptosis analysis by Annexin V-FITC/PI assay

The HDFCs of S-ODN group, AS-ODN group and untrasfected

group were cultured in DMEM (no FBS) at 4 h before

transfection procedure. After 48 or 72 h of transfection the

cells were harvested by trypsin-EDTA treatment, washed with

cold PBS and stained with PI and fluorescein isothiocyanate

(FITC)-conjugated Annexin V using an Annexin V-FITC

Apoptosis Detection Kit I (Becton Dickinson). Annexin V-FITC

identifies cells in early apoptosis by detecting externalised

phosphatidylserine, and PI identifies cells that have lost

plasma membrane integrity (i.e., necrotic or late apoptotic

cells). The cells were resuspended in 50 mL of 1� binding buffer

supplemented with 5 mL of Annexin V-FITC and 10 mL of PI,

and kept at room temperature in the dark for 15 min according

to the manufacturer’s instructions. Following the addition of

450 mL of 1� binding buffer, the stained cells were kept on ice

and subjected to fluorescence-activated cell sorter (FACS)

analysis using a FACSCalibur flow cytometer (BD Biosciences,

San Diego, CA, USA) with Cell Quest software (BD Biosciences).

We measured the FITC fluorescence between 515 and 545 nm

and the PI fluorescence between 564 and 606 nm.

2.8. Alkaline phosphatase (ALP) activity detection

After 48 and 72 h of transfection respectively, the cells of all

five groups were washed three times with 0.1 M PBS and 50 mL

of cold 10 mM Tris–HCl buffer (pH 7.4) containing 0.1%

TritonX-100 that was added before incubation at 4 8C over-

night. ALP substrate solution (100 mL) containing 2 mM MgCl2and 16 mM p-nitrophenyl phosphate was then mixed with

each sample. After incubation at 37 8C for 30 min, the reaction

was stopped by adding 50 mL of 0.2 M NaOH and the liberated

p-nitrophenol was measured spectrophotometrically at

410 nm. Each experiment was repeated for five times and

the data were presented as the means � S.D.

2.9. Effects of ADAM28 AS-ODN on HDFCs expressingECM proteins

Immunocytochemical (LsABC kit, DAKO, USA) staining and

image analysis were performed as described above. Polyclonal

antibody against bone sialoprotein (BSP), osteopontin (OPN)

Fig. 1 – The source identification of HDFCs. (a) The fourth passa

Immunocytochemical staining indicated that vimentin was stro

cytoplasms and cytomembranes of HDFCs respectively. Scale b

and dentin sialophosphoprotein (DSPP) (Santa Cruz, CA, USA)

were at 1:150 dilution. PAb against type I/III collagen (Santa

Cruz, CA, USA) was at 1:300 dilution. Control experiments

were carried out by replacing the primary antibody with non-

immune rabbit serum. The average greyscales (means � S.D.)

of each group were subjected to statistical analysis.

2.10. Statistical analysis

Results were analysed and expressed as the means � standard

deviations (S.D.). Statistical significance was assessed by the

Student-Newman-Keuls (SNK) test from SPSS-Windows ver-

sion 13.0 programme (SPSS Inc., Chicago, IL, USA). Differences

were considered significant at P < 0.01.

3. Results

3.1. The morphology and source identification of HDFCs

HDFCs presented fusiform, spindle or polygon-shaped in

primary cultures. However, most of the cells were fusiform-

shaped after they were continuously subcultured. The fourth

passage HDFCs which showed fusiform fibroblastic appear-

ance were used in this study. The HDFCs nucleus were shown

in round, orbicular-ovate and anachromasis. The nucleoli

were clear and cytoplasms were filled with particles (Fig. 1a).

The shapes and growth property of HDFCs from four groups

after transfection had no distinct difference with those before

transfection, minority of HDFCs appeared suspension or death

and attached total cellular score kept at 4 � 106/mL.

Immunocytochemical staining showed that vimentin was

positive in cytoplasms and cytomembranes of HDFCs (Fig. 1b),

while cytokeratin was negative in HDFCs (Fig. 1c) which

demonstrated that all isolated HDFCs were derived from the

mesenchyme with no contamination from dental epithelial

cells.

3.2. The identification and detections after transfection ofADAM28 eukaryotic expression plasmid

RT-PCR results showed that a 2327 bp fragment corresponding

to total length of ADAM28 coding region (amino acids 48–2375)

was cloned and displayed in 1% agarose gel electrophoresis.

Two fragments about 5428 and 2327 bp were generated after

pcDNA3.1(+)-ADAM28 was digested with EcoR I and Not I, and a

ge HDFCs with fusiform fibroblastic shape.

ngly positive (b) while cytokeratin was negative (c) in

ar = 20 mm.

Fig. 2 – The identification and expression of ADAM28 eukaryotic plasmid. (a) A 2327 bp fragment was obtained by PCR

identification after pcDNA3.1(+)-ADAM28 was digested with EcoR I and Not I. Lanes: 1, PCR product; 2, digestion

identification with EcoR I and Not I; 3, PCR identification; M, 15,000 bp DNA ladder. (b) ADAM28 mRNA was overexpressed in

eukaryotic plasmid group as shown by immunofluorescence assay. Scale bar = 50 mm. (c) RT-PCR detection showed that a

specific bright band of 2327 bp was more generously expressed in eukaryotic plasmid group than in pcDNA3.1(+) group and

untransfected group. Lanes: 1, untransfected group; 2, pcDNA3.1(+) group; 3, eukaryotic plasmid group; M, 15,000 bp DNA

ladder. (d) The quantification analysis of relative greyscale among three groups was calculated by the Delta CT method with

ADAM28/GAPDH, which was normalised to the GAPDH controls. The relative greyscale value of eukaryotic plasmid group

was higher than that of other groups. Significant difference was found between eukaryotic plasmid group and other two

groups (*P < 0.01). (e) Western blot detection displayed a more clear protein band of 35.3 kDa and elevated ADAM28

expression in eukaryotic plasmid group than in other two groups. Lanes: 1, untransfected group; 2, pcDNA3.1(+) group; 3,

eukaryotic plasmid group; M, protein ladder. (f) The quantitative analysis of relative greyscale revealed that the relative

greyscale value of eukaryotic plasmid group indicated by ADAM28/GAPDH was notably higher than that of other groups.

There was significant difference between eukaryotic plasmid group and other two groups (*P < 0.01). Quantitative data are

expressed as the means W S.D. *P < 0.01 versus other groups.

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5 839

2327 bp fragment was obtained by PCR identification (Fig. 2a).

BLAST search against sequence database indicated that the

insert of 2327 bp was human ADAM28 whose open reading

frame was complete and no mutation was found.

Immunofluorescence assay indicated that strongly green

fluorescence appeared in pcDNA3.1(+)-ADAM28 group

whereas weaker fluorescence was found in other two

groups (Fig. 2b). RT-PCR detection and relative greyscale

analysis showed that the corresponding brightness of

GAPDH (900 bp) band for each group was almost the same,

whereas a specific bright band of 2327 bp representative of

ADAM28 mRNA was more generously expressed in eukar-

yotic plasmid group than in pcDNA3.1(+) group and

untransfected group (Fig. 2c and d). Significant difference

was found between eukaryotic plasmid group and other two

groups (*P < 0.01). Western blot detection and relative

greyscale analysis revealed that the GAPDH (36 kDa) band

for each group was almost the same, but a more clear

protein band of 35.3 kDa (ADAM28) was found in eukaryotic

plasmid group than in other two groups (Fig. 2e and f). There

was significant difference between eukaryotic plasmid

group and other two groups (*P < 0.01). These results

confirmed the high efficiency of the present transfection

system and ensured the premise of gene manipulations for

further analysis of biological characteristics of HDFCs.

3.3. Transfection efficiency and inhibitive effect ofADAM28 AS-ODN on HDFCs

Strongly green fluorescence was found in cytoplasms and

nucleoli of the AS-ODN and S-ODN group under microscope.

Transfection efficiency of the AS-ODN and S-ODN group was

87%, 84% respectively, and there was no significant difference

between two groups (Fig. 3a).

Immunocytochemical staining showed that the expression

level of ADAM28 in HDFCs was significantly decreased after AS-

ODN transfection and significant difference was found between

groups (Fig. 3b and c). RT-PCR result indicated that the

corresponding brightness of GAPDH (900 bp) band for each

group was almost the same, whereas ADAM28 (255 bp) band of

the AS-ODN group was distinctly weaker than that of theS-ODN

and untransfected groups. Statistical analysis showed that

ADAM28 expression level of the AS-ODN group was lower than

that of other groups, and significant difference was detected

between groups (*P < 0.01) (Fig. 3d and e). Western blot analysis

revealed that a 35.3 kDa protein band in the AS-ODN group was

distinctly weaker than that of other two groups and there was

significant difference between groups (*P < 0.01) (Fig. 3f and g).

Quantitative analyses of these differences suggested that the

designated AS-ODN had satisfactory blocking effects on

ADAM28 expressions in HDFCs.

Fig. 3 – Transfection efficiency and blocking effect of ADAM28 AS-ODN on HDFCs. (a) Strongly green fluorescence was found

in cytoplasms and nucleoli of the AS-ODN and S-ODN group. There was no significant difference between two groups. Scale

bar = 50 mm (b) Immunocytochemical staining indicated that the expression level of ADAM28 in AS-ODN group was notably

decreased than that of other two groups. The expression level became inversely proportionate to average greyscale. Scale

bar = 50 mm (c) Quantitative analysis of average greyscale revealed that significant difference was found between AS-ODN

group and other groups (*P < 0.05). (d) RT-PCR detection indicated that the corresponding brightness of GAPDH band for each

group was almost the same, whereas ADAM28 band of the AS-ODN group was distinctly weaker than that of the S-ODN and

untransfected groups. Lanes: 1, S-ODN group; 2, AS-ODN group; 3, untransfected group. (e) The quantitative analysis of RT-

PCR bands displayed that the relative greyscale value of AS-ODN group indicated by ADAM28/GAPDH was markedly lower

than that of other groups and significant difference was found between AS-ODN group and other groups (*P < 0.01). (f)

Western blot revealed that a 35.3 kDa protein band in AS-ODN group was markedly weaker than that of other two groups.

(g) The quantitative analysis of Western blot bands showed that the relative greyscale value of AS-ODN group was lower

than that of other groups. There was significant difference between groups (*P < 0.01). Quantitative data are expressed as

the means W S.D. *P < 0.01 versus other groups

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3.4. Effects of ADAM28 eukaryotic plasmid and AS-ODNon biological property of HDFCs

The MTT assay was performed to study whether ADAM28

could affect cell proliferation in vitro. As displayed in Fig. 4a,

the eukaryotic plasmid-treated HDFCs exhibited conspicu-

ously higher optical density (OD) value than the control cells

at 48 and 72 h, while in ADAM28 AS-ODN group cell

proliferation was markedly inhibited at a statistically

significant level. The results indicated that ADAM28 appeared

Fig. 4 – Effects of altered ADAM28 gene expression on proliferation, apoptosis and differentiation activity of HDFCs. (a) The

eukaryotic plasmid-treated HDFCs exhibited a significantly higher optical density (OD) value than the control cells at 48 and

72 h, while in ADAM28 AS-ODN group cell proliferation was markedly inhibited at a statistically significant level. Data are

presented as the means W S.D. from five groups.*P < 0.01 versus other groups (b) Cell cycle distributions analysed by flow

cytometry revealed that eukaryotic plasmid-treated HDFCs presented a conspicuously higher percentage of cells in S/G2/M

phases(*P < 0.01), while transfection of ADAM28 AS-ODN resulted in markedly lower percentage of cells in S/G2/M phases

when compared with other groups (*P < 0.01). (c and d) Cell apoptosis percentage analysis indicated that there were more

numerous apoptotic cells in AS-ODN group than in other groups. (e) ALP activity assay showed that ADAM28 eukaryotic

plasmid-transfected HDFCs produced the lowest level of ALP activity, while in AS-ODN group ALP activity remained at a

higher level after 48 or 72 h culture. Data are shown as the means W S.D. from five groups.*P < 0.01 versus other groups (f)

untransfected and AS-ODN transfected cells stained positively for cytoplasmic OPN and type III collagen, whereas negative

staining was detected in S-ODN group. Scale bar = 50 mm (g) Quantitative analysis showed that the average greyscale value

of AS-ODN group expressing OPN and type III collagen was lower than that of other groups and significant difference was

detected between S-ODN group and other groups (*P < 0.05). Data are shown as the means W S.D. from five groups.

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5 841

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5842

to have a positive regulatory effect on the proliferation of

HDFCs.

Cell cycle distributions analysed by flow cytometry

revealed that eukaryotic plasmid-treated HDFCs presented a

notably higher percentage of cells in S/G2/M phases (Fig. 4b,

*P < 0.01), while transfection of ADAM28 AS-ODN resulted in

markedly lower percentage of cells in S/G2/M phases when

compared with other groups (Fig. 4b, *P < 0.01), demonstrating

that the deletion of ADAM28 could inhibit the proliferation of

HDFCs.

To detect whether this effect was related to the changes in

cell survival, cell apoptosis assay was carried out which

suggested that there were constitutively more numerous

apoptotic cells in ADAM28 AS-ODN group (Fig. 4c and d).

In contrast to the cell proliferation assay, ALP activity

analysis displayed a reverse trend that ADAM28 eukaryotic

plasmid-transfected HDFCs produced the lowest value of ALP

absorbance while AS-ODN group kept in a higher level after 48

or 72 h culture (Fig. 4e). The data showed that ADAM28 could

significantly inhibit ALP activity of HDFCs.

3.5. Effects of ADAM28 AS-ODN on HDFCs expressingECM proteins

Immunocytochemical staining results indicated that OPN and

type III collagen were stained positively in cytoplasms of

untransfected and AS-ODN transfected cells, whereas nega-

tive staining was shown in S-ODN transfected cells (Fig. 4f).

Quantitative analysis revealed that significant difference was

found between S-ODN group and other groups (Fig. 4g,

*P < 0.05). These results indicated that ADAM28 could sig-

nificantly inhibit the expressions of OPN and type III collagen

and present negative correlation with them.

4. Discussion

The multiple functions possessed by ADAM family members

suggest many potential roles for ADAM28. Our recent

researches about human ADAM28 continuous expressions

in tooth germ, ameloblasts, dental papilla cells and dental

follicle cells suggested that ADAM28 was involved in crown

and root morphogenesis process.10 The initiation of root

formation is determined by Hertwig’s epithelial root sheath

(HERS), which induces the differentiation of odontoblasts

forming root dentine.11 Moreover HERS and dental follicle cells

were stained positively from bell stage, which suggested that

ADAM28 could play a major role in maintaining the structure

of the dental follicle and the inducement to cervical loop, both

as a substrate and as a reservoir of paracrine molecules.10

Consistent with above reports, our present study demon-

strates convincingly the expression and localisation of

ADAM28 in HDFCs and further validated that ADAM28 might

function as a potential regulator involved in the proliferation,

specific differentiation and apoptosis of HDFCs.

In the present study, ADAM28 eukaryotic expression

system and antisense oligodeoxynucleotides (AS-ODN)

technique with reverse function were applied successfully.

ADAM28 could be correctly transcribed, translated and

expressed after transfected into HDFCs. Antisense oligonu-

cleotide biotechnology provides an efficient and specific

method for inhibiting gene expression and exploring gene

function, and multiple application of antisense inhibitory

experimental strategies have been leveraged to facilitate the

understanding of the signals involved in inductive epithe-

lial–mesenchymal interactions during tooth morphogen-

esis.23–25

Then we demonstrated that ADAM28 had an up-regula-

tive effect on the proliferation of HDFCs by MTT assay and

cell cycle detection. Cell proliferative kinetics is an essential

feature during tooth morphogenesis since high cell prolif-

erative activity is desirable for rapid increase in volume of

the tooth germ and tooth cusp formation.26 Cell cycle

detection revealed that more cells accumulated in S phase

(DNA synthesis phase) of pcDNA3.1(+)-ADAM28 group,

which resulted in the elevation of total PI value (cell

proliferation index, S + G2 + M) distinguishable from those

of control groups. The critical step required for the

proliferation of cells is their transition from cell cycle arrest

to entry into the active phase of the cell cycle through the G1/

S restriction point, thus the fast transition can enhance

higher rates of cell proliferation.27 Above results would argue

that ADAM28 facilitate the HDFCs proliferation by promoting

the transition process from G1 to S phase, further accelerat-

ing DNA reproduction and protein synthesis of HDFCs. This

is probably due to the process that ADAM28 eukaryotic

plasmid might enhance its catalytic activity of metallopro-

teinase, cleave matrix protein and rebuild tissue structure to

regulate cell proliferation liveness.28 Moreover, ADAM28

comprises an EGF-like domain, which may possess certain

functions of EGF such as the stimulation or maintenance of

undifferentiated cell proliferation during embryonic devel-

opment.29 Thus, our results verified beyond doubt the causal

relationship between ADAM28 and HDFCs proliferation.-

Bone-specific alkaline phosphatase is regarded as a non-

collagenous protein secreted by osteoblasts that is essential

for bone mineralisation such as cementum and considered

to be a highly specific marker of osteoblast differentiation

function.30,31 Of particular importance in mineralisation are

the high concentrations of ALP found in matrix vesicles,

which in dentine are the sites of initial crystal formation,

proposed to be involved in the formation of mantle dentine

and also in maturation of the extracellular matrix.32 In

addition, during dentinogenesis, the extracellular matrix

synthesised by the odontoblasts coincides with their

expression level of ALP.33 Accordingly, ALP activity is

generally used for an important reference index in detecting

odontogenic mesenchymal cytodifferentiation and matrix

mineralisation.34

In this study, it seemed that the overexpression of ADAM28

inhibited the differentiation of HDFCs while the use of

ADAM28 AS-ODN produced opposite effect, as indicated by

ALP activity. These evidences suggested that ADAM28, as an

important regulator between the epithelium and mesench-

yme,10 might participate in early formation, proliferation and

differentiation start of HDFCs.10

Cytodifferentiation expressed by ALP activity, and cell

proliferation evaluated by the MTT assay and cell cycle

analysis, coincidentally demonstrated that ADAM28 could

promote HDFCs proliferation during the process of differ-

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5 843

entiation inhibition, which was in accordance with the

previous concept that proliferation and differentiation are

inversely correlated with each other due to the existence of

dual-function regulators participating in controlling both the

processes.35 Therefore, it is reasonably proposed that some

components of ADAM28 could act not only as promoters for

the proliferation of HDFCs but also as inhibitors for cytodiffer-

entiation.

Apoptosis is a key process in the embryological develop-

ment of the tooth, periodontal ligament and supporting oral

tissue in the progression of oral disease, bone resorption,

immunological response and inflammation, and in wound

healing and certain pharmacological effects.36 Cell apoptosis

and proliferation are interrelated and interact with each

other in tooth development, and they both participate in

sculpturing the shape of teeth. Previous work displayed that

epithelial–mesenchymal tissue interactions prevent apop-

tosis. Moreover, apoptosis represents an important process

in teeth morphogenesis and remodelling during tooth

development. Understanding of apoptosis regulation in

the vestigial tooth primordia can help to elucidate the

mechanism of their suppression during evolution and to

identify factors essential for tooth survival.37 Hence, apop-

tosis may be a general mechanism for the silencing of

embryonic signalling centres including odontogenesis signal

transmission.38

In this study, it was shown that transfection of AS-ODN

could notably induce apoptosis of HDFCs which strongly

suggested ADAM28 might actively participate in the negative-

feedback regulation mechanism of apoptotic event in the

mesenchyme during tooth morphogenesis. Nevertheless,

there is much to be learned about its relationship and

interaction with other signalling molecules relevant to

apoptosis in tooth development. Of particular interest has

been the application of antisense inhibitory experimental

strategies to advance a suggested causal relationship between

signal transduction and inductive epithelial–mesenchymal

interactions.23

The extracellular matrix (ECM) is a biologically active tissue

composed of a complex mixture of macromolecules, that in

addition to serving a structural function, also profoundly

affect the cellular physiology of an organism.39 In addition, the

ECM relays multiple signals from the cell microenvironment to

direct proliferation and differentiation during tissue develop-

ment,40 and plays significant roles in tissue physiology

through interaction with cells and interstitial fluid transport.

These roles include regulating cell morphology, growth and

intercellular signaling.41,42 Cell adhesion, migration, prolifera-

tion and differentiation are examples of biological processes

influenced by the composition and structural organisation of

surrounding extracellular matrices.43

It is well known that osteopontin, bone sialoprotein (BSP)

and type III collagen are the ECM molecules which are

thought to be closely related to cytodifferentiation, matrix

secretion, mineralisation and bone formation, OPN and BSP

have also been regarded as critical markers for osteogenic

differentiation.44 Bone sialoprotein and osteopontin (OPN)

are two major mineralised tissue-specific, non-collagenous

matrix glycoproteins that are almost expressed exclusively

in bone and other mineralised connective tissues, such as

dentine, cementum, alveolar bone and calcified cartilage

tissue.45–47 Subsequent studies have demonstrated that BSP

mRNA expression is associated with the onset of miner-

alisation and is essentially restricted to differentiated

osteoblasts, osteoclasts, cementoblasts, odontoblasts, ame-

loblasts and hypertrophic chondrocytes.48,49 OPN gene

expression with several proposed functions has been

associated with cell transformation and metastases in vivo

and in vitro.50,51 In bone formation studied in vitro, OPN is

expressed before the formation of a mineralised collagenous

matrix52 but at much higher concentrations after bone

formation, indicating a prominent role for OPN in both the

formation and resorption of bone.53 This divergent pattern

of OPN/BSP expression could be an important determinant

for the different characteristics of these two types of bone

metastasis, which is consistent with the proposed role of

OPN in differentiation and activation of osteoclasts and of

BSP as a stimulator of bone mineralisation.44 Thus, OPN and

BSP are believed to serve a vital role in osteogenic

differentiation, formation and remodelling of the miner-

alised tissue matrix.54

Type I and Type III collagen are regarded as important

factors mediating chemotaxis, cell attachment and migration.

They probably serve as developmental signals, which may be

critical to the regulation of interactions between periodontal

fibroblasts and the root surfaces, and thus influence the

regeneration and repair of the periodontium.54 Moreover, type

III collagen is present in reticular fibres which provide

elasticity to tissues and the characteristics that type III

collagen imparts to tissues in vivo make it a worthwhile

molecule to study for tissue engineering applications includ-

ing odontogenic tissue and cell regeneration.26

As mentioned above, the fact that DF cells finally generate

the three types of periodontal structure in a concomitant

manner renders it as a good model for study of proliferation,

differentiation and mineralisation process.7,54 Accordingly,

OPN, BSP and type III collagen were used for evaluation of

osteogenic differentiation and matrix formation in HDFCs in

this study. The fact that the expressions of OPN and type III

collagen were significantly enhanced due to transfection of

ADAM28 AS-ODN disclosed that the abolition of ADAM28

promoted lineage-specific differentiation of HDFCs. Maybe

there exists a negative correlation between ADAM28 and OPN/

type III collagen. As mentioned above, ADAM28 protein

contains a transmembrane domain, a cytoplasmic domain

and an EGF-like domain, which including components of the

extracellular matrix and thus might play an important role in

signalling transduction, intracellular protein maturation, or

localisation to sites of activity.16

In a word, this study provides comprehensive evidence

about expressions of ADAM28 in HDFCs and its influence on

biological features of HDFCs at transcription, translation and

protein levels, which demonstrate ADAM28, as a novel

regulator, might actively manipulate proliferation, differen-

tiation and apoptosis of HDFCs by interacting with multiple

signal molecules including ECM and other growth factors.

These data will not only be beneficial for the better prehension

and utilisation of HDFCs for regenerative dental therapy, but

also promising for developing feasible gene treatment against

congenital hypoplasia of tooth root.

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 8 3 5 – 8 4 5844

Acknowledgements

This study was supported by a grant from the Nature Science

Foundation of China (Project No. 30572046).

Competing interests: None declared.

Ethical approval: Ethical Approval was given by the Ethical

Committee on Human and Animal Research of General

Hospital of Chinese PLA.

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