Wu et al.

Int Chin J Dent 2004; 4: 15-18.

In situ hybridization analysis of USF1 mRNA expression inodontoblasts
Li-an Wu, DDS, PhD, Ling-ying Wen, DDS, Fu-sheng Yang, DDS, Yong Liu, DDS,and Xiao-jing Wang, DDS, PhD
Department of Pediatric Dentistry, College of Stomatology, Fourth Military Medical University,
Xi’an, P. R. China
Purpose: To detect and verify USF1 mRNA expression in odontoblasts.
Materials and Methods: T-USF1 clone prepared previously was used as template and the desired USF1cDNA segment was amplified by PCR with specific primers. The segment was labeled with digoxin as aprobe and in situ hybridization was performed on the mounting of odontoblasts.
Results: Evidently positive staining was shown in the cytoplasm of odontoblasts.
Conclusion: We verified the expression of USF1 mRNA in odontoblasts for the first time and provided
evidence for further research. (Int Chin J Dent 2004; 4: 15-18.)
Clinical Significance: The results indicate that USF1 might be involved in the maturation of odontoblasts
and the process of dentin formation.
Key Words: in-situ hybridization, odontoblast, mRNA, PCR, USF1.

Introduction
The proliferation, differentiation and mineralization of odontobalsts are the important components of
tooth development and this process is modulated by multiple signaling molecules to act synergetically andsuccessively.1 To investigate the functional adjustment of USF1 to odontoblasts, we use the probe preparedby ourselves to detect whether there is USF1 mRNA expression in odontoblasts in vitro.

Materials and Methods
Amplification of USF1 cDNA Probe by PCRWith the T-USF1 plasmids we constructed previously as template, Oligo software (Takara, Dalian, P. R.
China) was used to design the PCR primers adjunctively, the sequence of the upstream and downstream
primers were 5TGAAACCGAAGAGGGAACAG3 (281-300) and 5GGCATCGTCACTGGTGAAAG3(569-550), respectively. Twenty-five µL of PCR reaction system was made according to the kit instructionof BcaBestTM NA PCR Kit Ver 1.1 (Takara) which contained: 12.5 µL of 2xBca lst Buffer, 2.5 µL ofMgSO4, 0.125 µL of Bca-optimized Taq, 1.0 µL of upstream primer, 1.0 µL of downstream primer, 0.5 µLof dNTP, 1.0 µL of plasmids template, and 6.375 µL of H2O. The segments of USF1cDNA were amplifiedby PCR and the reaction conditions were as follows: at 94˚C for 2 minutes, 94˚C for 30 s, 55˚C for 1minute, 72˚C for 1 minute, repeated in 35 cycles and then 72˚C for 10 minutes, the production of PCR wasmeasured, collected and quantified by electrophoresis through 18 g/L agarose gel.
The Labeling of USF1 cDNA ProbeFifteen µL of PCR production which contained 3.0 µg of target USF1 cDNA segments was labeled withdigoxin DNA according to the kit instruction, and stored below 20˚C for future use.

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Cell Culture and the Preparation of Cell Creeping Slides
MDPC-232 odontoblasts were cultured in a MEM (Gibco, Gaithersberg, MD, USA) with 10% fetal calf
serum and Hela cells were cultured in DMEM (Gibco) with 10% fetal calf serum. Confluent cells weredigested with trypsin and cell number was counted. At the density of 2x104/mL, cells were seeded in the24-well culture plate in which coverslips were placed beforehand, and 0.5 mL liquid was dropped to eachwell. Cells were cultured at 37˚C, 5% CO2 and saturated humidity for 48 hours, then washed with PBS,
and stabilized with icy methanol at below 20˚C for 15 minutes, the liquid was discarded and the coverslips
were dried for future use.
In Situ Hybridization
The cell creeping slides were placed in gradient ethanol and DEPC water, then in 0.2 mol/L hydrochloric
acid for 10 minutes, 5 mg/mL proteinase K for 3 minutes, 3% H2O2 for 30 minutes, washed with water anddried. The labeled USF1 cDNA probe was diluted with prehybridization solution to 10 mg/mL, and thenput in boiled water for 10 minutes and an ice bath at below 20˚C for 10 minutes. After that, it was dropped
homogeneously to the cell creeping slides, and put in wet box overnight. The following day, cell creeping
slides were washed while agitated repeatedly with 2xSSC, 1xSSC, 0.5xSSC and 0.2xSSC successively,
then with Buffer I for 5 minutes, repeated twice, and sealed with 2% goat serum for 30 minutes. Then it
was dropped evenly with ALP coupled anti-digoxin antibody, and put in wet box incubating for 2 hours at42˚C. After washed with Buffer I for 5 minutes, repeated three times and Buffer III for 1 minute once, thecell creeping slides was dropped with NBT/BCIP in wet box at 42˚C for staining, then washed with
distilled water to terminate the reaction, dehydrated and sealed. For the negative control, prehybridization
solution was used to substitute for the probe, and for the positive control, Hela cell creeping slides wereused.

Results
PCR Production
As we expected, after PCR amplification and gel electrophoresis, T-USF1 plasmids produced a specificamplication production which was the size of 289 bp (Fig. 1).

Fig. 1. Electrophoresis of USF1 cDNA segment.

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Int Chin J Dent 2004; 4: 15-18.

Fig. 2. USF1 mRNA expression in odontoblasts.

Fig. 3. USF1 mRNA expression in Hela cells.

In Situ Hybridization
There were positive blue-purple particles or balls in the cytoplasm of odontoblasts (arrow), but nostaining in the nuclei (Fig. 2). There was no staining for negative control cells. For the positive control
Hela cells, there was blue-purple staining in the cytoplasm (Fig. 3).

Discussion
Odontoblasts are a kind of terminal cells that are differentiated from dental papilla cells, and they cansecret dentin matrix and promote its mineralization. Therefore, their dysfunction can lead to the defect ofdentin formation and tooth dysplasia.

Studies in recent years have confirmed that the proliferation,mineralization and extracellular matrix secretion of odontoblasts were modulated by the synergetic actionof multiple hormones, extracellular growth factors and intracellular transcriptional factors.3
USF1 is a member of transcriptional factor bHLH-zip basic helix-loop-helix-leucine zipper family which
was first purified partially form the nuclear extract of Hela cells by Sawadogo et al. in 1985. Moreover, it
was found that the addition of USF1 could increase 10 to 20 times of the transcriptional activity ofadenovirus MLP (major late promoter).4 Subsequent studies successfully cloned the whole long cDNA
sequence which encodes the human being 43kD USF1 and the whole long cDNA sequence which encodes
the mouse USF1, and ascertained that the USF1 gene locates in human being chromosome 1 q22-q23, andthe USF1 gene of mouse consists of 10 exons and 9 introns, with each exon coding concrete functional
domain.5 Recent findings indicate that in addition to modulate MLP function, USF1 extensively takes partin the modulation of other tissue-specific gene transcription such as BMP-4, TGFb-2, CyclinB1 andcollagen etc, which is mediated by E-box (the core sequence is 5’CANNTG 3’), thus to control the cellproliferation and cell cycles.6,7 Furthermore, it was found in an animal experiment that though USF1-nullmice were viable, they had behavioral abnormalities, among the double mutants of USF1 and USF2 genes,
mice were embryonic lethal, suggesting that USF proteins are essential in embryonic development.8
Though some data were accumulated about USF1, there is limited data in the literature regarding
biological function of USF1, especially the relationship between USF1 and tooth development was notreported domesticly and overseas. In the present study, PCR primers were designed according to USF1
cDNA sequence published in Genebank and confirmed to be highly specific by Blast analysis. With17

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Int Chin J Dent 2004; 4: 15-18.

T-USF1 clone made previously as template, desired USF1 segment was prepared by PCR and labeled asprobe, and then USF1 mRNA expression in odontoblasts MDPC-23 was demonstrated by in situ
hybridization. Combination with the previous data about USF1, our present study indicates that USF1
probably plays a role in the modulation of growth, proliferation, differentiation and secretion ofodontoblasts, which also provides experimental data for the possibility and necessity of exploring thebiological function of USF1further with reverse nucleotide block or mutant transfection methods.
Acknowledgment
We thank Doctor Wenxi He of our Oral Medicine Laboratory for his excellent technical assistance in cell culture.

References
1. Narayanan K, Srinivas R, Ramachandran A, Hao J, Quinn B, George A. Differentiation of embryonic mesenchymal
cells to odontoblast-like cells by overexpression of dentin matrix protein 1. Proc Natl Acad Sci USA 2001; 98: 4516-21.
2. Sun ZL, Fang DN, Wu XY, et al. Expression of dentin sialoprotein (DSP) and other molecular determinants by a newcell line from dental papillae, MDPC-23. Connect Tissue Res 1998; 37: 251-61.
3. Bronckers AL, Engelse MA, Cavender A, Gaikwad J, D'Souza RN. Cell-specific patterns of Cbfa1 mRNA and protein
expression in postnatal murine dental tissues. Mech Dev 2001; 101: 255-8.
4. Sawadogo M, Roeder RG. Interaction of a gene-specific transcription factor with the adenovirus major late promoter
upstream of the TATA box region. Cell 1985; 43: 165-75.
5. Henrion AA, Vaulont S, Raymondjean M, Kahn A. Mouse USF1 gene cloning: comparative organization within thec-myc gene family. Mamm Genome 1996; 7: 803-9.
6. Kingsley-Kallesen ML, Kelly D, Rizzino A. Transcriptional regulation of the transforming growth factor-beta2 promoter
by cAMP-responsive element-binding protein (CREB) and activating transcription factor-1 (ATF-1) is modulated byprotein kinases and the coactivators p300 and CREB-binding protein. J Biol Chem 1999; 274: 34020-8.
7. Ebara S, Kawasaki S, Nakamura I, et al. Transcriptional regulation of the mBMP-4 gene through an E-box in the5'-flanking promoter region involving USF. Biochem Biophys Res Commun 1997; 240: 136-41.
8. Sirito M, Lin Q, Deng JM, Behringer RR, Sawadogo M. Overlapping roles and asymmetrical cross-regulation of the USF
proteins in mice. Proc Natl Acad Sci USA 1998; 95: 3758-63.

Reprint request to:
Dr. Li-an Wu
Department of Pediatric Dentistry, College of Stomatology, Fourth Military Medical University
Xi’an 710032, P. R. China
E-mail: peosh@fmmu.edu.cn
Received October 2, 2003. Revised November 10, 2003. Accepted December 16, 2003.
Copyright ©2004 by the Editorial Council of the International Chinese Journal of Dentistry.

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