Wu et al.

Int Chin J Dent 2007; 7: 59-63.

Upstream stimulatory factor 1 regulates OPNmRNA expression inodontoblasts
Li-an Wu, DDS, PhD, Ling-ying Wen, DDS, Fu-sheng Yang, DDS, and Xiao-jing Wang,DDS, PhD
Department of Pediatric Dentistry, College of Stomatology, The Fourth Military Medical University, Xi’an,P. R. China
Purpose: To detect the effects of upstream stimulatory factor 1 (USF1) on the expression of osteopontin (OPN)
in odontoblasts, and explore its biological functions in tooth development.
Materials and Methods: Odontoblasts MDPC-23 were cultured and stably transfected with PCMV-USF1 orA-USF expression plasmids. The mountings of odontoblast coverslips in each group were prepared and total
RNA was extracted. Immunofluorescence staining was performed with specific anti-USF1 and anti-HA tagantibodies. Semi-quantitative RT-PCR was carried out to detect the expression of OPN and β-actin in eachgroup. Gray value of OPN and β-actin in each group was calculated and statistically analyzed.
Results: Clones of stable PCMV-USF1 and A-USF plasmids transfection were achieved. Positive staining of
HA was shown in the cytoplasm of odontoblasts in A-USF transfection group. Compared with the control,
PCMV-USF1 transfection group appeared stronger staining. Electrophoresis of semi-quantitative RT-PCR
showed that OPN was upregulated in PCMV-USF1 transfection group, while it was downregulated in A-USF
transfection group.
Conclusion: USF1 could regulate the expression of OPN in odontblasts, which could be blocked partially by
A-USF. (Int Chin J Dent 2007; 7: 59-63.)
Key Words: immunofluorescence staining, odontoblast, OPN, RT-PCR, USF1.

IntroductionOsteopontin (OPN) is an important component in dentinal matrix and involved in matrix mineralization, celltransformation, and bone absorption.

Upstream stimulatory factors 1 (USF1) belongs to the eucaryotic

evolutionary conserved basic-Helix-Loop-Helix-Leucine Zipper transcription factor family, can activate thetranscription of multiple growth and metabolism related genes, and regulate cell proliferation anddifferentiation.1 In previous researches,2,3 we cloned the specific coding sequence of USF1 from the tooth germof 5-day postnatally balb/c mouse and detected the expression of USF1mRNA in an odontoblastic cell line
MDPC-23. This experiment was aimed to detect the effects of USF1 on OPN mRNA expression in MDPC-23
by gene transfection and semi-quantitative PCR, and explore the biological function of USF1 during toothdevelopment.

Materials and Methods
Cell culture
Odontoblasts MDPC-23 were cultured in aMEM medium (Gibco, Grand Island, NY, USA) with 10% fetal
calf serum.
Plasmid transformation, extraction and quantitation
DH5a competent cells were transformed by PCMV-USF1 and A-USF plasmids, which contained
anti-Neomycin genes both but HA tag only in A-USF, were kindly gifted by Prof. Michele Sawadogo and Dr.
Charles Vinson respectively. After spread and culture, blue-white screening was carried out. Positive clones
were chosen to expand culture, and plasmids were extracted by plasmid extraction kit (Clonetech, Palo Alto, CA,USA) and dissolved in TE solution. Four µL were taken out and detected by ultraviolet spectrophotometer todetermine the purity and contents.

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Gene transfection and screening
Odontoblasts were transfected with PCMV-USF1 and A-USF plasmids with Lipofectamine 2000 according tothe instruction. Twenty-four hours after transfection, cells were passaged to a 60 mm flat plate at the ratio of
1:10. Three hundred µg/mL G418 was added to the cell culture medium one day after passage. Cell culture
medium and G418 were replaced everyday. After 10 days of screening, all cells in the control died, then G418
was reduced to 150 µg/mL until resistant clones emerged. Filter paper method was used to transfer the resistantclones to a 24 well culture plate and macro-culture was progressed further. Then cell creeping slides and total
RNA extraction were carried out as previously described.2,3
Immunofluorescence staining
In order to confirm the integration and expression of transfected plasmids by screened clones, the following
immunofluorescence staining was undertaken on the cell creeping slides respectively according to the kit
instruction: anti-USF1 staining in USF1 transfection group (antibody dilution 1:150), anti-HA staining inA-USF1 transfection group (antibody dilution 1:200). The secondary antibody was fluorescence-conjugatedgoat anti-rabbit-Texas Red (dilution 1:500). After incubation with the secondary antibody at 4˚C for 6 to 8 hours,
cell creeping slides were rinsed with PBS, mounted with glycerine containing anti-quench agents, and examinedwith fluorescence microscopic scope.
Semi-quantitative PCR
PCR primers of OPN were designed according to mouse OPNcDNA sequence (AF5157081) in Genebank.
Theforward

andbackward

5’ACTTGTGGCTCTGATGTTCC3’primers(437bp),were5’TCACCATTCGGATGAGTCTG3'andandthewereprimersofβ-actin

5’TGGTGGGTATGGGTCAGAAGGACTC3’ and 5’CATGGCTGGGGTGTTGAAGGTCTCA3’ (265bp).
Blast analysis revealed that both primers were specific. Reverse transcription PCR was progressed as described
previously with the same amount of RNA (171 ng each).3 PCR condition of OPN was 94˚C for 5 minutes, then94˚C for 30 s, 55˚C for 30 s, 72˚C for 1 minute, repeated 27 cycles and finally 72˚C for 7 minutes. PCR
condition of β-actin was 93˚C for 5 minutes, then entered the 33 cycles as 93˚C for 1 minute, 56˚C for 30 s, and72˚C for 1 minute, and finally stayed at 72˚C for 7 minutes.
Statistical analysis
The gray scale value of electrophoresis bands in each PCR reaction was measured by Labworks software; theration of OPN/β-actin in each group was calculated and compared by statistic software SPSS 11.0.

Results
Purity and contents of plasmids
The A260/A280 ratio of PCMV-USF1 and A-USF plasmids was 1.96 and 1.87, which indicated high purity
and suitable for gene transfection. The final concentration of both plasmids was adjustment to 0.3 µg/µL.
Stable transfection and clone screening
After MDPC-23 odontoblastic cells were transfected with the plasmids, both grew in good condition. The day
after adding G418, cell death began. Up to the 10th day, cells in untransfected group all died, while there
remained some scattered cells in transfected groups. Then maintaining screening at 150 µg/µL for another twoweeks until resistant cell clones emerged (Figs. 1 and 2).

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Fig. 1.
Fig. 2.

Int Chin J Dent 2007; 7: 59-63.

1
Clone stably transfected with PCMV-USF1 plasmids (left, x20).
Clone stably transfected with A-USF plasmids (right, x20).

2

Immunohistochemistry
Immunofluorescence staining showed that, compared with the control, anti-USF1 staining in USF1
transfection group was enhanced (Figs. 3 and 4), and that anti-HA staining was only positive in A-USF
transfection group (Fig. 5), indicating the success of stable transfection and integration of both plasmids.

Figs. 3-5.

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Staining of USF1 in MDPC-23 (left, x40), staining of USF1 in stable USF1 transfection group(center, x40), and staining of HA in A-USF transfection group (right, x40).

5

RT-PCR
Gel electrophoresis displayed that there was only a specific band at 437bp and 265 bp in each group,consistent with what we expected. The band brightness of β-actin in each group was equal, indicating the sameamount of template added to RT-PCR.

Compared with the control, brightness of OPN band in USF1

transfection group was brighter, but darker in A-USF transfection group (Fig. 6). Mearued by Labworks
software, the relative ratio of gray scale between OPN and β-actin in USF1, A-USF transfection group, andcontrol was 60.33±4.51%, 229.33±7.09%, and 110.00±15.62% respectively, which differed significantly fromeach other via statistical analysis.

437bp
265bpM123M12613Fig. 6.
Electrophoresis of RT-PCR for
OPN and β-actin.
M,Marker
Track 1, USF1 transcription group
Track 2, A-USF transcription group
Track 3, Control

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Int Chin J Dent 2007; 7: 59-63.

Discussion
OPN, a prominent component of the extracellular matrix of mineralized tissues in teeth, is a secretedglycosylated phosphoprotein containing specific sequences of ARG-GLY-ASP that can adsorb hydroxyapatite,form bridges between cells and minerals in the matrix, and mediate cell adhesion and migration. OPN was first
separated from bone matrix, and then detected in a variety of other tissues. It was found to be implicated inmatrix mineralization, cell transformation and bone matrix absorption, and have very tight relationship with teeth.
Wei et al.4 detected the expression of OPN in rat tooth development via immunohistochemical staining method,and found that OPN presented in ameloblasts only on 1d postnatal rats and expressed in both ameloblasts andodontoblasts in the ensuing 1-6 week-old rats, indicating that OPN plays an important role in the development
and mineralization of rat tooth. Zhang et al.5 observed the persistent OPN expression in odontoblasts andreparative dentin within 10 days after direct pulp capping, and presumed that OPN may be concerned with theformation of reparative dentin. In addition, OPN is involved in tooth root development and tooth absorption
process.6
The expression of OPN is tissue-specific and regulated by a variety of exogenous factors, growth factors, andtranscripton factors. Ohishi et al.7 noticed that retinoic acid could stimulate osteopontin synthesis by cultured
dental pulp cells in a dose-dependent manner, and 6.2 fold increases were observed at the concentrations of 10-6mol/L.

High glucose also enhances the expression of osteopontin in aortic smooth muscle cells at thetranscriptional level through the activation of protein kinase C-dependant pathway.8 While genetic structure ofOPN analyzed, several transcription factor recognition sites were identified, such as TATA-box, E-box, andGC-box in the promoter region. Transient transfection and gel-shift analyses of osteopontin promoter indicatedthat a region between -123 and +66 bp was crucial in the regulation of osteopontin expression, and that USF1
could specifically bind the E-box between -101 and -96 bp to be involved in the regulation of osteopontin
expression on vascular smooth muscle cells.9 An extensive deletion analysis of the 5'-flanking region of rat OPN
gene revealed that a high glucose response element was present within -112 and -62 bp of the OPN promoter
including an E-box and a GC-rich region. Mutation of the E-box or the GC-rich region resulted in at least 30%
loss of high glucose induced promoter activity.10 Bidder et al.11 confirmed further that OPN gene transcription
was regulated by USF and AP1 in aortic vascular smooth muscle cells, and that glucose up-regulated both AP1and USF DNA binding and transactivation activities. However, whether USF1 in odontoblasts could also
regulate the expression of OPN and mediate the effect of high-glucose, retinoic acid and other external factors on
tooth development is still unknown.
PCMV-USF1 and A-USF plasmids could code for full-length USF1 and A-USF protein. A-USF is a USF
dominant-negative mutant.12 In this construct, the basic region of the bHLH-zip domain of USF1 was replacedwith an acidic sequence, which greatly stabilizes the heterodimers between A-USF and USF. Consequently,
A-USF is a very efficient inhibitor of USF DNA binding and could effectively block the transcriptional control
activity of USF1. In this research, odontoblast MDPC-23 was cultured; stably trasfected with PCMV-USF1 andA-USF plasmids, then the variance of OPNmRNA expression in each group was detected by semi-quantitative
RT-PCR and compared statistically. The results showed that there was OPNmRNA expression in MDPC-23
odontoblasts, and that compared with the control, OPN mRNA was upregulated in PCMV-USF1 transfectiongroup, but downregulated in A-USF transfection group.

Thus it indicated that USF1 could regulate theexpression of OPN in odontblasts, which could be blocked partially by A-USF. These results provide evidence62

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for further researches on USF1 function in the process of odontoblasts maturation and dentin formation, andindicate that USF1 might be implicated in dentin matrix secretion and mineralization during tooth development.

References
1. Corre S, Galibert MD. Upstream stimulating factors: highly versatile stress-responsive transcription factors. Pigment Cell Res
2005; 18: 337-48.
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2002; 12: 588-90.
3. Wu LA, Wen LY, Yang FS, Liu Y, Wang XJ. In situ hybridization analysis of USF1 mRNA expression in odontoblasts. Int Chin J
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4. Wei SS, Cao CF, Meng HX. Expression of sialoprotein and osteopontin in rat tooth development. Chin J Stomatol 2002; 27:47-9.
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dentinogenesis in vivo. Chin J Dent Res 2000; 3: 38-43.
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7. Ohishi M, Horibem, Ikedo D, et al. Effect of retinoid acid on osteopontin expression in rat clonal dental pulp cells. J Endod1999; 25: 683-5.
8. Takemoto M, Yokote K, Yamazaki M, et al. Enhanced expression of osteopontin by high glucose in cultured rat aortic smoothmuscle cells. Biochem Biophy Res Comm 1999; 258: 722-6.
9. Malyankar UM, Hanson R, Schwartz SM, Ridall AL, Giachelli CM. Upstream stimulatory factor 1 regulates osteopontin
expression in smooth muscle cell. Exp Cell Res 1999; 250: 535-47.
10. Asaumi S, Takemoto M, Yokotek, et al. Identification and characterization of high glucose and glucosamine responsive
element in the rat osteopontin promoter. J Diabetes Complications 2003; 17: 34-8.
11. Bidder M, Shao JS, Charlton-Kachigian N, Loewy AP, Semenkovich CF, Towler DA. Osteopontin transcription in aortic
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12. QyangY, Luo X, Lu T, et al. Cell-type-dependent activity of the ubiquitous transcription factor USF in cellular proliferation andtranscriptional activation. Mol Cell Biol 1999; 19: 1508-17.

Correspondence to:
Dr. Li-an Wu
Department of Pediatric Dentistry, College of Stomatology, The Fourth Military Medical University
145 West Changle Road, Xi’an, 710032, P. R. China
Fax: +086-29-83223047
E-mail: ailingcun@yahoo.com.cn
Received June 24, 2007. Accepted August 13, 2007.
Copyright ©2007 by the International Chinese Journal of Dentistry.

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