Elsevier

Archives of Oral Biology

Volume 50, Issue 2, February 2005, Pages 227-236
Archives of Oral Biology

Altered gene expression in human cleidocranial dysplasia dental pulp cells

https://doi.org/10.1016/j.archoralbio.2004.10.014Get rights and content

Summary

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterised by defects of bone and tooth development. The dental manifestations in CCD patients include supernumerary teeth, delayed tooth eruption, tooth hypoplasia and absence of cellular cementum formation. This disorder is associated with mutations in the osteoblast-specific transcription factor Runx2. To identify morphological and molecular alterations associated with CCD dental tissues, human primary dental pulp cell cultures were established from age- and sex-matched CCD and normal patients. Dental pulp cells were compared for general morphology, proliferation rates, and gene expression profiles using cDNA microarray technology. CCD pulp cells were about four-fold larger than normal cells, however the normal pulp proliferation rates were two- and three-fold greater at time points tested than the CCD cells. Of the 226 genes analysed by blot microarray, 18.6% displayed significant differences at least two-fold in expression levels. This includes 25 genes (11.1%) that were up-regulated, while 17 (7.5%) that were down-regulated in the CCD cells as compared to the normal cells. Expression of selected genes was further verified by quantitative real-time polymerase chain reaction (qRT-PCR). Comparison between the CDD and normal cells revealed that gene expression of cytokines and growth factors, such as leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and transforming growth factor beta receptor II (TGF-βRII) and vascular endothelial growth factor B (VEGFB) were higher while bone morphogenetic protein 2 (BMP2) was lower in the CCD cells. Furthermore, potential Runx2 binding sites were found in all putative target gene promoters. This study suggests that in addition to bone and tooth cell differentiation, Runx2 may be involved in controlling cell growth during tooth development.

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.

References (60)

  • K. Tsuji et al.

    Expression of the PEBP2alphaA/AML3/CBFA1 gene is regulated by BMP4/7 heterodimer and its overexpression suppresses type I collagen and osteocalcin gene expression in osteoblastic and nonosteoblastic mesenchymal cells

    Bone

    (1998)
  • T. Aberg et al.

    Runx2 mediates FGF signaling from epithelium to mesenchyme during tooth morphogenesis

    Dev Biol

    (2004)
  • G. Xiao et al.

    Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2

    J Biol Chem

    (2002)
  • B. Lutterbach et al.

    A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia

    J Biol Chem

    (2000)
  • M. Goldberg et al.

    Pulpo-dentinal complex revisited

    J Dent

    (1995)
  • S.R. Nalabolu et al.

    Genes in a 220-kb region spanning the TNF cluster in human MHC

    Genomics

    (1996)
  • B. Olofsson et al.

    Genomic organization of the mouse and human genes for vascular endothelial growth factor B (VEGF-B) and characterization of second splice isoform

    J Biol Chem

    (1996)
  • O. Kozawa et al.

    Leukemia inhibitory factor enhances bFGF-induced IL-6 synthesis in osteoblasts: involvement of JAK2/STAT3

    Cell Signal

    (2002)
  • B. Lee et al.

    Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF/CBFA1 in cledocranial dysplasia

    Nat Genet

    (1997)
  • G. Zhou et al.

    CBFA1 mutation analysis and functional correlation with phenotypic variability in cleidocranial dysplasia

    Hum Mol Genet

    (1999)
  • R.N. D'Souza et al.

    Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice

    Development

    (1999)
  • P. Ducy et al.

    A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development

    Genes Dev

    (1999)
  • C.R. Chung et al.

    Micro-CT evaluation of tooth, calvaria and mechanical stress-induced tooth movement in adult Runx2/Cbfa1 heterozygous knock-out mice

    J Med Dent Sci

    (2004)
  • C. Banerjee et al.

    An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene

    Proc Natl Acad Sci USA

    (1996)
  • M. Kundu et al.

    Cbfbeta interacts with Runx2 and has a critical role in bone development

    Nat Genet

    (2002)
  • C.A. Yoshida et al.

    Core-binding factor beta interacts with Runx2 and is required for skeletal development

    Nat Genet

    (2002)
  • A. Javed et al.

    Runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins

    Mol Cell Biol

    (2001)
  • G.L. Barnes et al.

    Osteoblast-related transcription factors Runx2 (Cbfa1/AML3) and MSX2 mediate the expression of bone sialoprotein in human metastatic breast cancer cells

    Cancer Res

    (2003)
  • S. Chen et al.

    Spatial expression of Cbfa1/Runx2 isoforms in teeth and characterization of binding sites in the DSPP gene

    Connect Tissue Res

    (2002)
  • M.H. Lee et al.

    Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation

    J Cell Biochem

    (1999)
  • Cited by (30)

    • Tgfbr2 is required in osterix expressing cells for postnatal skeletal development

      2017, Bone
      Citation Excerpt :

      Transforming growth factor-β (TGFβ) is part of a superfamily of proteins that mediate a wide range of biological activities including proliferation, differentiation, and formation of the extracellular matrix [12–15]. Disruption of TGFβ signaling can cause skeletal and connective tissue disorders, including Marfan syndrome, Camurati-Engelmann disease, Cleidocranial Dysplasia and Loeys Dietz Syndrome [16–25]. TGFβ ligands initiate signaling by binding to the TGFβ type II receptor (Tgfbr2), which then recruits the TGFβ type I receptor (Tgfbr1) and consequently forms a heterotetrameric complex with two Tgfbr2 receptors and two Tgfbr1 receptors.

    • High-throughput Gene and Protein Expression Analysis in Pulp Biologic Research: Review

      2010, Journal of Endodontics
      Citation Excerpt :

      In vitro studies allow strictly regulated conditions, in which the environmental causes for expression differences are eliminated. Chen et al (60) used cDNA microarray containing probes for cytokines and cytokine receptors to study the differential gene expression between pulp cell cultures established from cleidocranial dysplasia patients and healthy controls and found markedly different expression of genes involved in cell growth and signaling. Liu et al (34) used oligonucleotide-based microarray to identify the genes associated with dental pulp stem cell mineralization, detecting up-regulation of the osteogenesis-related genes, the genes related to collagen metabolism, and the TGF-β related genes simultaneously with increased alkaline phosphatase activity.

    • Increased levels of CK19 mRNA in oral squamous cell carcinoma tissue detected by relative quantification with real-time polymerase chain reaction

      2006, Archives of Oral Biology
      Citation Excerpt :

      With advances in molecular technology, especially real-time reverse transcriptase polymerase chain reaction (RT-PCR), quantitative detection of target nucleic acids, including DNA and RNA, has become possible. Using real-time PCR, both absolute and relative quantifications have been shown to be sensitive and specific for target gene detection.12–17 However, relative quantification appears to have several advantages for detection; i.e. relative quantification may be easier to perform than absolute quantification because a standard curve is not required; normalizing to an endogenous reference gene can provide a method to correct the results for differing amounts of input RNA, which is particularly attractive when it is not practical to measure the amount of input RNA.16,17

    • Dental Pulp Stem Cells

      2006, Methods in Enzymology
      Citation Excerpt :

      Researchers have recognized that dental pulp contains ex vivo‐expandable cells called dental pulp cells. These cells express osteogenic markers, such as alkaline phosphatase, type I collagen, bone sialoprotein, osteocalcin, osteopontin, transforming growth factor β(TGF‐β), and bone morphogenetic proteins (BMPs) (Chen et al., 2005; Kuo et al., 1992; Nakashima et al., 1994; Pavasant et al., 2003; Shiba et al., 1998). They also respond to the induction of BMP, fibroblast growth factor 2 (FGF‐2), matrix extracellular phosphoglycoprotein (MEPE), and TGF‐β by undergoing osteogenic differentiation (Alliot‐Licht et al., 2005; Dobie et al., 2002; Iohara et al., 2004; Liu et al., 2005; Nakao et al., 2004; Saito et al., 2004; Unda et al., 2000).

    View all citing articles on Scopus
    View full text