Runx2, osx, and dspp in tooth development

Abstract

The transcription factors Runx2 and Osx are necessary for osteoblast and odontoblast differentiation, while Dspp is important for odontoblast differentiation. The relationship among Runx2, Osx, and Dspp during tooth and craniofacial bone development remains unknown. In this study, we hypothesized that the roles of Runx2 and Osx in the regulation of osteoblast and odontoblast lineages may be independent of one another. The results showed that Runx2 expression overlapped with Osx in dental and osteogenic mesenchyme from E12 to E16. At the later stages, from E18 to PN14, Runx2 and Osx expressions remained intense in alveolar bone osteoblasts. However, Runx2 expression was down-regulated, whereas Osx expression was clearly seen in odontoblasts. At later stages, Dspp transcription was weakly present in osteoblasts, but strong in odontoblasts where Osx was highly expressed. In mouse odontoblast-like cells, Osx overexpression increased Dspp transcription. Analysis of these data suggests differential biological functions of Runx2, Osx, and Dspp during odontogenesis and osteogenesis.

Figure 1.

Runx2, Osx, and Dspp expression patterns in developing teeth and surrounding tissues from E14 to E18. Runx2 mRNA was clearly detected in dental and osteogenic mesenchyme from E14 to E16, but its expression was down-regulated in odontoblast, ameloblast, and dental pulp cells at E18 of the mandibular incisor and first molar. Osx expression mostly overlapped with Runx2 expression from E14-16. At E18, its expression was intense in ameloblasts, odontoblasts, and dental pulp cells. Dspp transcripts were absent at E14, but its transcript signals were slightly detected in dental and osteogenic mesenchyme at E16. At E18, Dspp transcripts were evident in odontoblasts and pre-ameloblasts in the mandibular incisor and first molar. am, ameloblasts; b, alveolar bone; c, Meckel’s cartilage; de, dental epithelium; df, dental follicle; dp, dental papilla; mc, mesenchymal condensates; od, odontoblasts; p, dental pulp cells.

Figure 2.

Runx2, Osx, and Dspp expression patterns in developing teeth and surrounding tissues at post-natal days (PNs) ranged from PN 1 to PN 10. Runx2 mRNA was detected in osteogenic mesenchyme and down-regulated in ameloblasts, odontoblasts, and dental pulp cells. In contrast, Osx expression was seen in odontoblasts and dental pulp cells. Dspp mRNA was strongly expressed in odontoblasts and weakly expressed in ameloblasts at post-natal (PN) days 5 and 10 during tooth development.

Figure 3.

Osx and Dspp expression in mouse odontoblast-like cells. Immortalized mouse odontoblast-like cells (MO6-G3) were photographed by light microscopy (A,E). Expression of Osx and Dspp proteins in MO6-G3 cells was analyzed by immunostaining with primary anti-Dsp or anti-Osx antibody (B and F, respectively). Cells were stained with Hoechst for the nucleus (C,G). Panels D and H are composites of B-C and F-G, respectively. Dspp signal was detected in the cytoplasm in the mouse odontoblast-like cells. Osx protein was expressed in both the nucleus and cytoplasm.

Figure 4.

Effect of Osx on Dspp and Runx2 expressions. Mouse odontoblast-like and osteoblast cells were transfected with either Osx or empty expression plasmid as a control. After 48-hour transfection, total RNA was used for qRT-PCR and semi-quantitative PCR. The primer sequences used for qRT-PCR were as follows: Dspp, forward 5′-AACTCTGTGGCTGTGCCTCT-3′ and reverse 5′-TATTGACTCGGAGCCATTCC-3′; Runx2, forward 5′-AGTGCTCTAACCACAGTCCATGCA-3′ and reverse 5′-TACAAACCATACCCAAGTACCTGTTT-3′; Osx, forward 5′-ATGGCGTCCTCTCTGCTTGA-3′ and reverse 5′-TTGAGAAGGGAGCTGGGTAG-3′; and cyclophilin, forward 5′-GGTGACTTCACACGCCATAA-3′ and reverse 5′-CATGGCCTCCACAATATTCA-3′. The mRNA expression without Osx overexpression (empty vector transfectant) in MO6-G3 or ST2 cells is designated as a 1.0-fold increase. Expression levels of Runx2, Osx, and Dspp mRNAs in MO6-G3 or ST2 cells with Osx transfectants are represented as fold-changes in relation to the control. All data are represented by mean ± SD from 3 independent experiments performed in triplicate. *P values lower than 0.05 were considered statistically significant. For semi-quantitative PCR, a pair of primers of the glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene was used as an internal positive control, as follows: forward 5′-CCATGGAGAAGGCCGGG-3′ and reverse 5′-CAAAGTCATGGATGACC-3′. The primers of Runx2 and Osx are described above. Quantitative RT-PCR was used to detect mRNA expression levels of Dspp (A), Runx2 (B), and Osx(C) in MO6-G3 or ST2 cells with or without Osx transfection. D. Semi-quantitative PCR analysis of Runx2, Osx, and GADPH mRNA expressions in MO6-G3 and ST2 cells transfected with or without Osx expression vector.