Expression of Runx2 and Its Signaling Partners Tcf7, Fgfr1/2 in Cleidocranial Dysplasia

RUNX2 is a member of the PEBP2/CBF transcription factors family controlling the expression of genes whose products are essential for bone formation. Mutations in the RUNX2 gene may be associated with cleidocranial dysplasia (CCD), a rare skeletal disease characterized by stature aberrations, delayed closure of the cranial su-tures, hypoplastic or aplastic clavicles, and multiple dental abnormalities. As RUNX2 is involved in many signaling pathways, we hypothesize that CCD may be associated with their changes. We determined the expression of RUNX2 and its signaling partners TCF7, involved in canonical Wnt signaling, and fibroblast growth factor receptors, FGFR1 and FGFR2 in periodontum of CCD patients and control individuals. We did not observe any differences between the level of RUNX2, TCF7 and FGFR1/2 mRNA, determined by real-time PCR, in CDD patients and controls. Therefore, RUNX2 signaling pathways with their partners TCF7 and FGFR1/2 may not be involved in CCD pathogenesis.


INTRODUCTION
The human RUNX2 gene (also designated Cbfa1, PEBP2A1, Osf2 or AML3) is a member of the mammalian Runx (runt-related transcription factor x) gene family, including also Run2 and Run3.All of these genes encode transcription factors homologous to the Drosophila runt protein whose products form heterodimers with transcriptional co-activator core-binding factor β, Cbfβ, binding the same DNA consensus sequence 5'-PyGPyG-GTPy-3' with runt-homologous DNA binding domains (Jonason et al., 2009).RUNX2 is essential for proper tooth morphogenesis and it interacts with Runx3 to regulate maturation and proliferation of chondrocytes (D 'Souza et al., 1999;Wuelling & Vortkamp, 2011).RUNX2 transcription can be initiated either from an upstream promoter or a promoter within the third intron and its primary transcript may be alternatively spliced, which results in three known RUNX2 transcripts.Moreover, several post-translational modifications can be imposed on the polypeptide product of RUNX2 expression (Shui et al., 2003).Multiple signaling pathways are involved in the regulation of the RUNX2 gene expression at multiple levels.These include growth hormones, cy-tokines and hormones, playing an important role in signal transduction regulating spatial and temporal expression of RUNX2 (Komori, 2011;Nishimura et al., 2012).Aberrant expression of RUNX2 is important, because RUNX2 activity should be tightly regulated temporally and spatially to properly control the rate of bone formation (Nishimura et al., 2012).It is also a key target of mechanical stimulation in human periodontal ligament osteoblastic cells (Ziros et al., 2002).In general, the role of RUNX2 in tooth formation seems to be of a special significance because of its essential function for the later stages of this process and differentiation of mesenchymal cells to osteoblasts (Ducy et al., 1997).
An important role of RUNX2 in osteoblast differentiation is underlined by a network of protein-protein interactions with essential role of RUNX, SP7, FGF and Wnt signaling (Komori, 2011).
Mutations in the RUNX2 gene may play an important role in the pathogenesis of cleidocranial dysplasia (CCD, OMIM 119600, cleidocranial dysostosis), a skeletal disorder associated with dysplatic clavicles, patent sutures and multiple supernumerary teeth (Jensen & Kreiborg, 1990;Lee et al., 1997;Bergwitz et al., 2001).Disturbed pattern of dentition is probably the most pronounced hallmark of CCD.The absence of functional RUNX2 may result in the inhibition of the development of molars and incisors at the late bud and bell stage, respectively (D 'Souza et al., 1999;Aberg et al., 2004).However, besides a direct action of RUNX2 on the pattering of dentition, this protein is involved in a complex network of interactions with many signaling proteins and transcriptional factors (Komori, 2011).This interaction involves the Wnt/betacatenin, TGF-beta/BMP, FGF, Notch and Hedgehog signaling pathways, and RUNX2, Osterix, ATF4, TAZ, and NFATc1 transcriptional factors, but they represent only part of all proteins involved (Deng et al., 2008).Therefore, in assessing the connection between disturbed RUNX2 expression and pathological phenotypic trait, such as CCD, all these interactions should be taken into account.Due to high number of proteins involved in these interactions it is rather difficult to study them in a single cycle of experiments.In this work we investigated the level of mRNA expression of RUNX2 and three genes interacting with it -TCF7, FGFR1 and FGFR2, in teeth of CCD patients and controls.
The TCF7 (Transcription factor 7 (T-Cell Specific, HMG-Box) also known as Tcf1) gene is located at 5q31.1 and encodes a transcriptional activator that plays an important role in lymphocyte differentiation.It is a Wnt/βcatenin target gene and may play a role in cementoblast/ osteoblast differentiation (Silverio et al., 2012).Wnt signaling may increase the expression of RUNX2 through binding of TCF7 to its promoter (Gaur et al., 2005).On the other hand, TCF7 was induced by RUNX2 in the process of chondrocyte maturation and proliferation (Mikasa et al., 2012).No information on association of this gene with CCD is available.
Fibroblast growth factors (FGFs) are a family of 22 protein ligands acting in paracrine or endocrine fashion (Goetz & Mohamadi, 2013).They are bound by their receptors, encoded by the FGFR1-4 genes, which dimerize upon binding and signal through their tyrosine kinase activity.FGF signaling is important for development and tissue homeostasis.Mutations in the FGFR1 and FGFR2 genes play a role in the pathogenesis of craniosynostosis and other skeletal growth disorders (Morriss-Kay & Wilkie, 2005).FGF/FGFR signaling stimulates expression, transcriptional and DNA-binding activity of RUNX2 (Kim et al., 2003;Lu et al., 2012) and is important in controlling of self-renewal and differentiation of dental epithelial stem cells (Chang et al., 2013).The FGF signaling is essential in mammalian tooth development (Li et al., 2014).No direct association on FGF signaling and CCD has been reported although a putative functional relationship between type 2 craniosynthosis and CCD was suggested (Rice et al., 2003).
Therefore, RUNX2 may be involved in mutual regulation of TCF7 and FGFR1/FGFR2 in the process of osteoblast differentiation in tooth development (Fig. 1).It is possible that this network may be disturbed in pathologies associated with tooth development, including CCD.We speculate that the level of the expression of genes encoding these proteins may be associated with CCD.To test this hypothesis we checked the expres-sion of the RUNX2, TCF7, FGFR1 and FGFR2 genes in teeth of CCD patients and controls.This study was approved by the Bioethics Committee of the Medical University of Lodz, Lodz, Poland (permit no.RNN/151/11/KE) and each patient gave an informed consent, which in the case of minors was signed by their parents.All individuals were personally interviewed and 3 CDD cases were considered as hereditary, while the rest -as sporadic.

Patients
Reagents.RNeasy Mini Kit was obtained from Qiagen (Valencia, CA, USA), Maxima First Strand cDNA Synthesis Kit for RT-qPCR was purchased from Thermo Scientific (Northpoint Parkway, FL, USA), Tris, EDTA were obtained from Sigma (St. Louis, MO, USA).All other reagents were of the highest purity available.
RNA isolation.Total RNA was isolated with RNeasy Mini Kit from periodontum obtained by mechanical scraping from teeth extracted from 12 CCD patients and 15 control individuals without CCD.All samples were obtained following informed consent for genetic testing, but the tooth extraction itself was a routine procedure Expression was calculated according to the 2-∆C T formula with human β-actin as a reference gene.Each measurement was performed in duplicate, error bars denote S.E.M. RUNX2 signaling in cleidocranial dysplasia followed for clinical reasons.RNA samples were stored in the TE buffer (5 mM Tris/HCl, 0.1 mM EDTA, pH 8.5) at -20°C until use.
Gene expression and data analysis.cDNA was synthesized with Maxima First Strand cDNA Synthesis Kit.The TaqMan Gene Expression Assay was employed to analyze the expression of the RUNX2 (probe ID Hs00231692), TCF7 (Hs00175273), FGFR1 (Hs00915142) and FGFR2 (Hs01552918) genes in a thermal cycler CFX96™ Real-Time PCR Detection System (BIO-RAD Laboratories, Hercules, CA, USA).The gene for human β-actin (ACTB) was used as an internal control for all real time reverse transcription quantitative polymerase chain reactions (RT-qPCRs).The thermal conditions were: 10 min of polymerase activation at 95°C, followed by 40 cycles of 30 s denaturation at 95°C and 60 s annealing/extension at 60°C.Each sample was run in duplicate.Negative controls containing no DNA were included in each RT-qPCR run.The cycle threshold (C T ) values were calculated automatically by CFX96 Real-Time PCR Detection System software and the 2-∆C T method was used to calculate relative basal expression of mRNA of each gene normalized to the average mRNA, i.e. ∆C T was obtained by the subtraction of C T of ACTB mRNA from C T for each gene (Schmittgen & Livak, 2008).Data were analyzed by the Student's ttest for CCD patients and controls.

RESULTS AND DISCUSSION
We did not observe any difference (p>0.05) between the expression level of the RUNX2, TCF7, FGFR1 and FGRFR2 genes in CCD patients and controls (Fig. 1).
The RUNX2 gene plays an important role in the tooth formation and pattering through a complex network of signaling, including many protein-protein interactions.Our knowledge about all relationships within this network is far from completeness, which impedes molecular view of pathologies associated with impaired function of RUNX2.So far, CCD is the only abnormality associated with mutations in the RUNX2 gene.However, molecular mechanism on the pathway from mutated RUNX2 to the CCD phenotype is largely unknown.Moreover, about 30% of all CCD cases are not associated with RUNX2 mutations (Baumert et al., 2005;Zhang et al., 2010).Some information on this mechanism may be provided by comparing the level of expression of RUNX2 itself and genes whose products directly interact with it.In the present work we analyzed the expression of three such genes, TCF7, FGFR1 and FGFR2, which were not studied in CCD so far.Our results indicated that the signaling pathways of RUNX2 along with these three proteins may not be involved in CCD pathogenesis.
It was shown that a TCF regulatory element responsive to canonical WNT signaling is located in the RUNX2 promoter and TCF7 along with β-catenin is recruited to RUNX2 (Gaur et al., 2005).It was also shown in that report that coexpression of TCF7 with proteins of canonical WNT pathways resulted in overexpression of Runx2 in mouse.The conclusion from the data obtained in that research was that TCF7 with WNT signaling, activated the expression of Runx2 to control osteoblast differentiation and skeletal development.Therefore, disturbance in this signaling pathway may lead to disturbed osteogenesis, resulting also in anomalous tooth formation.However, this abnormality may not involve that observed in CCD, as our data suggest.
FGF/FGFR signaling may regulate RUNX2 expression and activity in several ways, including activation of protein kinase C (PKC) (Kim et al., 2003).RUNX2 has multiple phosphorylation sites and its phosphorylation may be associated with both inhibition and activation (Franceschi et al., 2003;Wang et al., 2004;Xiao et al., 2000).It was shown that FGFR2 upregulated the transactivating activity of exogenously overexpressed RUNX2, but inhibitors of PKC hampered this overexpression.Therefore, although PKC signaling may increase expression of RUNX2, which is usually associated with increase in the activity of RUNX2, this signaling pathway may also stimulate transactivation functions of RUNX2 by other mechanism(s) (Kim et al., 2006).FGF2 may mediate transactivation of RUNX2 by phosphorylation of its Ser247 residue.It was shown that RUNX2 did not exert direct effects on the promoter of the FGFR2 gene, but it inhibited the stimulatory effect of Twist1 on this promoter (Lu et al., 2012).
Our patient population was relatively small and so, our results have limited significance and this research should be continued to draw more robust conclusions.However, CCD is a rare disease (incidence about 1:200 000) and our results suggest that the interaction of RUNX2, with TCF7 and TGFR1,2 may not be involved in its pathogenesis, which is an important information in the context of contribution of RUNX2 to the etiology of this disease and its involvement in many signaling pathways.
and ethics.This study included 12 CCD patients (7 males and 5 females, age range 17-22) and 15 controls who were enrolled at the Department of Developmental Dentistry and Department of Orthodontics, Medical University of Lodz, Lodz, Poland in 2012-2014.The diagnosis of CCD was determined on the basis of clinical signs of the disease and supported by mutational analysis at the Department of Clinical Genetics, University of Lodz, Lodz, Poland.The only treatment the patients had undergone was extraction of supernumerary teeth.Control individuals were age-and sex-matched, they did not show signs of CCD and had tooth extraction performed for variousreasons, most often due to pain.

Figure 1 .
Figure 1.Relative mean mRNA expression of the RUNX2, TCF7 and FGFR1/2 genes in patients with cleidocranial dysplasia (n=12) and controls (n = 15).Expression was calculated according to the 2-∆C T formula with human β-actin as a reference gene.Each measurement was performed in duplicate, error bars denote S.E.M.