Altered gene expression in human cleidocranial dysplasia dental pulp cells
Introduction
Cleidocranial dysplasia (CCD) is an autosomal dominant human disease affecting both bone and tooth formation. The main symptoms of CCD patients are open fontanelles, hypoplasia or aplasia of the clavicles, a wide public symphysis, and short stature.1, 2, 3, 4 Additionally, dental disorders include supernumerary teeth, abnormal tooth eruption, tooth hypoplasia and lack of cellular cementum formation. CCD is associated with mutations of Runx2, a transcription factor essential for osteoblast and dental cell differentiation as well as bone and tooth formation.5 Mice with targeted disruption of Runx2 gene−/− show no osteoblast differentiation with a complete lack of bone formation and exhibit changes in early dental development.6, 7 Tooth germs are arrested at the cap/early bell stages with no odontoblast cytodifferentiation and dentin matrix formation.8 Recently, studies have demonstrated that Runx2 is also involved in fetal and postnatal growth of bone9 and postnatal formation of dental tissues and tooth eruption.10, 11
Runx2 (also known as Cbfa1, Osf 2, Pebp2aA or AML-3) belongs to the runt-domain gene family since its DNA binding domain shares a high degree of homology with the Drosophila pair-rule gene, runt.12 Runx2 specifically recognises a core DNA sequence and heterodimerises with the Cbfβ unit, a cofactor that enhances affinity of Runx2 to DNA.13, 14 Runx2 controls transcription of many bone- and tooth-related genes through its binding site.5, 15, 16, 17, 18, 19, 20, 21 In addition, Runx2 is regulated by bone morphogenetic proteins (BMPs)/transforming growth factor β (TGF-β) family22, 23, 24, 25 and other growth factors,8, 26 by the mitogen-activated protein kinase pathways,27 and by Smad proteins that are signal transductors.28, 29, 30 Besides being a key determinant of the osteoblast lineage and differentiation as well as bone formation, recent studies have shown that Runx2 regulates genes related to cell growth and controls osteoblast proliferation.31, 32, 33, 34, 35, 36 Dentin and bone share several characteristics at molecular levels and contain mineralised collagen matrices and non-collagenous proteins, such as osteocalcin (OC), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) and alkaline phosphatase (ALP). It is proposed that cell-matrix interactions shown to be crucial for osteoblast differentiation are also necessary for odontogenesis. Both odontoblast and dental pulp cells originate from the dental papilla mesenchyme derived from cranial neural crest cells. Several in vitro and in vivo studies have demonstrated that dental pulp cells are capable of differentiating into odontoblasts and producing a mineralising matrix, particularly during reparative dentinogenesis.37, 38 In situ hybridisation studies have shown differential expression of Runx2 isoforms (types I–II) in mouse developing teeth with expression of all forms in odontoblast and dental pulp cells.21
In this study, we analysed basic cell properties and broadly surveyed the gene expression profiles of human primary dental pulp cells isolated from a CCD versus normal patient. Over 200 human genes, cytokines and receptors involved in various biochemical pathways, were analysed as potential Runx2 downstream target genes using a microarray assay. Genes identified as potentially regulated by Runx2 were confirmed by qRT-PCR analysis and a bioinformatics approach through identification of putative Runx2 binding sites in their promoters.
Section snippets
Cell lines
Human primary normal (Runx2+/+) and CCD (Runx2+/−) dental pulp cells were established from extracted teeth of a CCD patient as well as a normal age- and sex- matched control with signed informed consent as previously outlined.39 The patient with CCD was an 11-year-old male and showed clavicular hypoplasia, short stature, low nasal bridge, and supernumerary teeth as described previously by our laboratory.40 The genotype of the CCD patient showed that arginine at position 225 was replaced with
Cell morphology and growth rates of Runx2+/+ and Runx2+/− dental pulp cells
We first observed the basic cell morphology of Runx2+/+ and Runx2+/− dental pulp cells using phase contrast microscopy (Fig. 1A). Compared to the normal pulp cells, Runx2+/− cells appear flat and larger. Comparison of cell volume measurements showed that the volume of the Runx2+/− cells was about four-fold greater than the Runx2+/+ cells (data not shown). In contrast, the cell proliferation rate of the normal pulp cells was two- and three-fold greater compared to that of the Runx2+/− cells at
Discussion
In the present study, we found that the osteoblast-specific transcription factor Runx2 contributes to the control of cell growth in human primary dental pulp cells. The phenotype and genotype of the patients with CCD used in this study have been described earlier40 and shared some common characteristics as reported by other groups.1, 2, 3, 4 These phenotypes include clavicular hypoplasia, short stature, low nasal bridge, and supernumerary teeth. A missense mutation at 225 (R225Q) within the
Acknowledgments
This work was supported by National Institute Dental and Craniofacial Research Grant DE 113221.
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