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research-article2015
JDRXXX10.1177/0022034514566655Journal of Dental ResearchAbnormal Differentiation of Dental Pulp Cells
Research Reports: Biological
Abnormal Differentiation of Dental Pulp Cells in Cleidocranial Dysplasia
Journal of Dental Research 2015, Vol. 94(4) 577–583 © International & American Associations for Dental Research 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0022034514566655 jdr.sagepub.com
W.J. Yan1,2*, C.Y. Zhang1*, X. Yang3, Z.N. Liu4, X.Z. Wang1, X.Y. Sun1, Y.X. Wang5, and S.G. Zheng1
Abstract Cleidocranial dysplasia (CCD) is a skeletal dysplasia caused by heterozygous mutations of RUNX2, a gene that is essential for the mineralization of bone and tooth. We isolated primary dental pulp cells from a 10-y-old patient and tested their proliferative capacity, alkaline phosphatase activity, and ability to form mineralized nodules, in comparison with those from 7 healthy children. All these measures were reduced in primary dental pulp cells from the CCD patient. The expression of the osteoblast/odontoblast-associated genes RUNX2, ALP, OCN, and DSPP was also found to be significantly decreased in the primary dental pulp cells of the CCD patient. The osteoclast-related markers TRAP, CTSK, CTR, and MMP9 were decreased in primary dental pulp cells cocultured with human peripheral blood mononuclear cells. Moreover, the expression of RANKL and the ratio of RANKL/OPG were both reduced in the cells from the CCD patient, indicating that the RUNX2 mutation interfered with the bone-remodeling pathway and decreased the capacity of primary dental pulp cells to support osteoclast differentiation. These effects may be partly responsible for the defects in tooth development and the retention of primary teeth that is typical of CCD. Keywords: cell biology, craniofacial anomalies, bone remodeling/regeneration, craniofacial biology/genetics, osteogenesis, osteoclasts
Introduction Cleidocranial dysplasia (CCD; MIM 119600) is an inherited autosomal-dominant bone disease that is characterized by hypoplasia of the clavicle and dental abnormalities (Cooper et al. 2001). The main systemic manifestations include delayed closure of fontanelle or persistently open fontanelle, widened suture and the sutural bone (Wormian bones), unilateral/bilateral clavicle hypoplasia or absence, short stature, and other skeletal anomalies (Mundlos 1999). Dental abnormalities mainly consist of primary teeth retention, delayed eruption of permanent dentition, and supernumerary teeth (Jensen and Kreiborg 1991; Shaikh and Shusterman 1998). It has been demonstrated that heterozygous mutation of the RUNX2 gene (synonyms: CBFA1, AML3, OSF2, PEBP2A) is responsible for CCD (Mundlos et al. 1997; Otto et al. 1997). This osteoblast-specific transcription factor, which is mapped to human chromosome 6p21, is involved in regulation of bone metabolism and plays an important role in osteoblast differentiation and osteoclast formation. It has been shown that RUNX2 specifically regulates the transcription and expression of some genes related to bone and tooth development, including osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (Col I), osteopontin (OPN), bone sialoprotein (BSP), and ameloblastin (AMBN; Ducy et al. 1997). Runx2 homozygous mutant mice exhibit lack of bone formation owing to maturational arrest of osteoblasts (Komori et al. 1997), while Runx2 heterozygous mutant mice exhibit specific characteristics of CCD, such as hypoplastic nasal bones and clavicles (Otto et al.
1997). RUNX2 also plays an important role in the differentiation of osteoclasts and odontoclasts through its regulation of the RANKL/RANK/OPG signaling pathway (Sato et al. 2008). RUNX2 binding elements have been found in the promoter region of OPG and RANKL (Aberg et al. 2004), indicating that the expression of OPG and RANKL could be directly regulated by RUNX2. 1
Department of Preventive Dentistry, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China 2 Department of Pediatric Dentistry, the First Division, Peking University School and Hospital of Stomatology, Xicheng District, Beijing, China 3 Department of Stomatology, Dongzhimen Hospital Beijing University of Chinese Medicine, Dongcheng District, Beijing, China 4 Department of Prosthodontics, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China 5 Central Laboratory, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China *First coauthors contributing equally to this article. A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. Corresponding Authors: S.G. Zheng, Department of Preventive Dentistry, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, China. Email: [email protected] Y.X. Wang, Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidan District, Beijing 100081, China. Email: [email protected]
578 The mechanism producing dental abnormalities in CCD patients is not fully understood. However, it is well known that primary dental pulp cells (DPCs) play an important role in the development and eruption of permanent successors (Yildirim et al. 2008); therefore, changes to the biological characteristics of primary DPCs could lead to some of the dental abnormalities associated with CCD. In this study, primary DPCs of a patient with CCD (a natural RUNX2 gene mutation model) were examined to see if their proliferation, mineralization, or differentiation behavior differed from those of healthy controls.
Materials and Methods Patient This study was approved by the Ethical Committee of Peking University School of Stomatology (approval No. PKUS SIRB2012004). A Chinese child aged 10 y old who was clinically and genetically diagnosed with CCD was examined with informed parental consent (Zhang et al. 2010).
Cell Culture Retained deciduous mandibular lateral incisors of the CCD patient were extracted; one third of the root length had been resorbed. Briefly, dental pulp was isolated, and 2 mm from the apical region was discarded; then, the remainder was digested in a mixture of collagenase type I (3 mg/mL; Sigma-Aldrich, MO, USA) and dispase (4 mg/mL; Sigma-Aldrich), and the single-cell suspensions were incubated in DMEM medium (Gibco, MO, USA) containing 10% fetal bovine serum (Hyclone, UT, USA). Cells were incubated at 37 °C in 5% CO2. The third to fifth passage of the primary DPCs was used for the experiments. Nine retained mandibular deciduous incisors from 7 unaffected children aged 6 to 8 y old were used as controls. The extracted incisors had a similar pattern of root resorption to those of the CCD patient.
Immunohistochemical Staining for Cell Identification Primary DPCs were plated on coverslips at 2 × 104 cells per well in a 24-well plate. When 70% confluency was reached, the cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 min and then permeabilized with 0.2% Triton X-100 in phosphate buffered saline for 10 min. Next, the cells were incubated overnight at 4 °C with primary antibodies against cytokeratin and vimentin (Zhongshan Bioengineering Co. Ltd., China). Then the procedure was performed according to the protocol of an SP immunohistochemical kit and a 3,3′-diaminobenzidine coloration kit (Zhongshan Bioengineering Co. Ltd.). Adipocytes and squamous carcinoma cells were used as positive controls for vimentin and keratin staining. The results were observed with a light microscope equipped with a camera (BX51 microscope and DP72 camera; Olympus, Japan).
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Cell Proliferation Assay Primary DPCs were plated in 96-well plates (2 × 103 cells per well) containing DMEM growth (basal) medium. After 12 h, a cell proliferation assay was performed with a Cell Counting Kit 8 (Sigma) according to the manufacturer’s instructions. Absorbance was measured at 450 nm with an ELx808 absorbance microplate reader (BioTeK, USA).
ALP and Alizarin Red Staining Primary DPCs were plated in 12-well plates at a density of 2 × 104 cells per well. At 80% confluency, the growth medium was replaced with osteogenic medium, composed of 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 10 nM L-ascorbic acid in growth medium. After 7 d, ALP staining was performed with an ALP histochemical staining kit (Cwbiotech, Beijing, China), and ALP activity was analyzed with an ALP activity assay kit (Jiancheng, Nanjing, China) according to the manufacturer’s instructions. Primary DPCs were seeded into 12-well plates (2 × 104 cells per well). After 24 h, they were treated with osteogenic medium and cultured for another 21 d. Then, they were fixed with 4% paraformaldehyde and stained with alizarin red solution (Sigma-Aldrich).
Quantitative Reverse Transcription Polymerase Chain Reaction Primary DPCs were seeded into 6-well plates (1.2 × 105 cells per well). At 80% confluency, the growth medium was replaced with osteogenic medium. After 7 d, the total RNA was extracted with Trizol (Invitrogen, CA, USA), and the RNA was reverse transcribed as previously described (Liu et al. 2013). The resultant cDNAs were amplified through the specific sets of primers for ALP, DSPP, OCN, and Col I. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used to normalize RNA expression levels. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed with a SYBR Green PCR kit (Roche Applied Science, IN, USA) according to the manufacturer’s protocol, and the results were assessed with 7500 Software 2.0.1 (Applied Biosystems, CA, USA).
Western-Blot Analysis Primary DPCs (4 × 105 cells) were seeded on 100-mm dishes. When 80% confluency was reached, the growth medium was replaced with osteogenic medium, and they were incubated in this for 7 d. Then the total protein was extracted with a protein lysis buffer (Applygen, Beijing, China). Protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal aliquots of total protein (40 μg) were electrophoresed in a 7.5% SDS-PAGE gel and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were incubated in 5% nonfat dry
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Abnormal Differentiation of Dental Pulp Cells milk for 1 h to block the nonspecific binding sites and incubated with primary antibodies against DSPP, RUNX2, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated anti-rabbit or anti-mouse IgG (Jackson Immuno Research, PA, USA) were then used, and immunoreactive bands were detected using a western enhanced chemiluminescence blotting kit (Applygen Technology Inc., Beijing, China).
Coculture of Primary DPCs and Human Peripheral Blood Mononuclear Cells Human peripheral blood from 2 healthy volunteers was layered on a Ficoll-Paque density gradient and centrifuged at 1800 rpm for 30 min. The white membrane layer between the FicollPaque and blood plasma was collected; this mainly consists of peripheral blood mononuclear cells (PBMCs). The primary DPCs were plated in 24-well plates (3 × 104 cells per well). When 60% confluency was reached, PBMCs were plated into the same wells at 3 × 106 cells per well. After 7 d, the mRNA of the cells was extracted, and real-time polymerase chain reaction was performed with a SYBR Green PCR kit to investigate the expression of the osteoclast-associated genes TRAP, CTSK, CTR, and MMP9. Then, the ratio of RANKL/OPG was measured for the primary DPCs.
Statistical Analysis Data are expressed as means ± SD. Differences between groups were determined by 1-way analysis of variance. The threshold for statistical significance was set at P < 0.05.
Results Clinical Manifestation of CCD in the Patient This patient is a family case whose father was also affected by CCD. The patient showed typical CCD features, including hypoplastic clavicles, dental abnormalities, and classic craniofacial features, such as insufficiently developed midface and prominent forehead (Appendix Fig.). Dental anomalies included retained deciduous teeth, delayed eruption of permanent teeth, and supernumerary teeth (Appendix Fig. C–F). Although this patient was already 10 y old, all the teeth in the mouth were deciduous except for the first permanent molars and the lower left incisor. In a previous study, we used mutation analysis to identify a heterozygous frame shift mutation in the RUNX2 gene of this patient (c.514delT, p.Ser172fs; Zhang et al. 2010); this confirmed the clinical diagnosis of CCD.
RUNX2 Mutation Decreased the Growth Rate of Primary DPCs Immunohistochemical staining was first performed to verify the source of the primary DPCs. The DPCs from the CCD
patient and normal control were positive for vimentin staining and negative for keratin staining, indicating that the isolated cells were mesenchymal cells (Fig. 1A). The morphology of the primary DPCs was spindle shaped, similar to fibroblasts for CCD (RUNX2+/m) and normal cells (RUNX2+/+; Fig. 1B). However, the growth rate was observed to be slower for CCD versus normal cells, becoming noticeable on day 3 (Fig. 1B). Quantitative analysis through a Cell Counting Kit 8 proliferation assay confirmed this decrease, with significantly lower cell numbers being found for days 2 to 10 (Fig. 1C).
RUNX2 Mutation Reduced the Mineralization Capacity of Primary DPCs ALP production, which is regulated by RUNX2, is one of the main indicators of mineralization and odontoblast differentiation (Trowbridge 2003). Enhanced ALP activity is considered an early marker of DPC differentiation and dentin formation (Tsukamoto et al. 1992). Therefore, the effect of the CCDassociated RUNX2 mutation on ALP expression was assessed. The basal level of ALP mRNA in the CCD patient’s DPCs was reduced by 79% compared with the normal control (Fig. 2A). A similar result was found after stimulation with osteogenic medium; 63% reduction versus the control (Fig. 2A). ALP staining was performed to investigate the effect of RUNX2 gene mutation on ALP expression at the protein level. ALP staining showed a lower ALP expression in the CCD patient (Fig. 2B). Quantitative analysis showed that ALP protein levels were reduced by approximately 30% in the CCD cells, under both basal and osteogenic conditions (Fig. 2C); this indicates reduced mineralization. Alizarin red staining was then performed to study the effect of RUNX2 mutation on the formation of mineralized nodules. In agreement with the ALP results, the number of mineralized nodules in the CCD cell cultures was noticeably reduced when compared with control cultures, after stimulation in osteogenic medium (Fig. 2D).
RUNX2 Mutation Reduced the Expression of Osteoblast/Odontoblast-associated Genes in Primary DPCs The expression of osteoblast/odontoblast-associated genes in the primary DPCs was analyzed. DSPP, OCN, and Col I were chosen for analysis because they are potential downstream targets of RUNX2 (Ducy et al. 1997; Chen et al. 2002). For primary DPCs from the CCD patient, RUNX2 mRNA levels were reduced about 60% in both basal and osteogenic media (Fig. 3A), and the mRNA levels of OCN and DSPP showed a similar pattern (Fig. 3B, C), whereas Col I levels remained unchanged (Fig. 3D). RUNX2 and DSPP protein levels were detected by Western blot. In agreement with the mRNA measurements, the expression of RUNX2 and DSPP protein was reduced in the CCD cells in both media (Fig. 3E).
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Figure 1. The proliferation of primary dental pulp cells (DPCs) was delayed by RUNX2 mutation. (A) Vimentin and keratin immunohistochemical analyses for primary DPCs from normal control and cleidocranial dysplasia (CCD) patient. ADPs, adipocytes; RUNX2+/+, normal control; RUNX2+/m, CCD patient; SCCs, squamous carcinoma cells. (B) Representative photomicrographs of primary DPCs from normal control and CCD patient at indicated time point after incubation. (C) Proliferation assay for primary DPCs from normal control and CCD patient at indicated time point after incubation. *P < 0.05. **P < 0.01.
RUNX2 Mutation Reduced the Expression of Osteoclast-related Genes and Modified Bone Remodeling Activity Osteoblasts and other mesenchyme-derived cells can induce differentiation of osteoclast precursors into osteoclasts (Fujikawa et al. 1996; Quinn et al. 1998), and DPCs have been shown to induce differentiation of PBMCs into osteoclasts (Uchiyama et al. 2009). Through a coculture composed of PBMCs and primary DPCs, the expression of osteoclastrelated genes was measured to assess the effect of CCD and normal primary DPCs on the differentiation of PBMCs into osteoclasts. The mRNA levels of TRAP, CTSK, CTR, and MMP9 were all reduced by the RUNX2 mutation in the primary DPCs from the CCD patient (Fig. 4A–D), indicating decreased osteoclast activity in the coculture. The RANKL/OPG signaling pathway plays an important role in the differentiation and maturation process of osteoclasts
(Suzuki et al. 2004). RANKL mRNA levels were reduced by 90% in the CCD case compared with the control (Fig. 4E). In addition, a 35% increase in OPG mRNA was found for the CCD DPCs (Fig. 4F). Accordingly, the ratio of RANKL/OPG was reduced by about 92% (Fig. 4G), indicating impairment of the RANKL/OPG signaling pathway when the function of RUNX2 is disturbed by heterozygous mutation.
Discussion Although there are numerous skeletal deficiencies in CCD patients, the dental disorder is most often their primary concern and major cause of reduced quality of life. The patient included in this study had typical features of CCD, such as hypoplastic clavicles and dental abnormalities. A heterozygous mutation in RUNX2 was detected in this patient, further confirming the causal role of RUNX2 mutation in CCD (Mundlos et al. 1997). To explain the typical dental anomalies of this patient, we
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hypothesized that the CCDassociated RUNX2 mutation may affect the mineralization capacity of the patient’s primary DPCs along with their ability to induce osteoclast differentiation. We found that ALP activity and mineralization of the primary DPCs were reduced by RUNX2 mutation. The expression of osteoblast/ odontoblast-related genes, including RUNX2, ALP, OCN, and DSPP, was significantly decreased in primary DPCs from the CCD patient. So, mineralization capacity of primary DPCs was reduced by the RUNX2 mutation. We found that the growth rate of primary DPCs from the CCD patient was slower than normal control. Figure 2. RUNX2 mutation reduced the alkaline phosphatase (ALP) activity and mineralization of This is consistent with a previous primary dental pulp cells (DPCs). DPCs from normal control (RUNX2+/+) and cleidocranial dysplasia study on DPCs (Xuan et al. 2010). (CCD) patient (RUNX2+/m) were isolated and cultured in osteogenic medium, which was composed of RUNX2 plays an important role in 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 10 nM L-ascorbic acid for 7 or 21 d. proliferation of chondrocytes (Hinoi (A) The ALP mRNA levels of normal and CCD patient DPCs were detected by quantitative reverse transcription polymerase chain reaction after incubating in osteogenic medium for 7 d. (B) The ALP et al. 2006); it is also involved in staining of DPCs from normal control and CCD patient was tested by ALP histochemical staining on regulating the expression of cell day 7. (C) ALP activity for DPCs from normal control and CCD patient was quantified on day 7. **P cycle–related genes, such as TGFB< 0.01. (D) Typical mineralized nodules stained with alizarin red for DPCs from normal control and CCD patient on day 21. RII, VEGFB, and BMP2 (Ji et al. 2001; Chen et al. 2005). However, Retention of the primary teeth and delayed eruption of perthe role of RUNX2 in proliferation of primary DPCs needs to be manent dentition are the most common oral manifestations in further investigated. CCD patients. The exfoliation of deciduous teeth is a complex As a transcription factor, RUNX2 plays an essential role in and orderly physiologic process, which involves a balance the development of bone and tooth (Ducy et al. 1997; D’Souza between osteoblast/odontoblast and osteoclast/odontoclast et al. 1999). Many related genes are regulated by RUNX2, activity. Periodontal ligament cells and dental follicle cells play such as OCN, ALP, Col I, OPN, BSP, and AMBN (Ducy et al. an important role in the process, and primary DPCs have also 1997). Among them, ALP and OCN are respectively considbeen shown to be involved. The odontoclastic resorption of ered early- and late-stage markers of DPC differentiation and coronal dentin takes place just before shedding (Sahara et al. dentin formation. DSPP is recognized as a specific marker of 1992). Primary DPCs can produce cytokines that induce cells of odontoblast differentiation, and there are RUNX2 binding sites the monocyte-macrophage lineage to become osteoclasts/odonin the regulatory elements of the mouse Dspp gene (Chen et al. toclasts. During physiologic resorption, RANKL and CSF-1 2002). Therefore, this study explored the effect of the CCDmRNA expression was shown to increase in primary DPCs associated RUNX2 mutation on the expression of ALP, OCN, (Yildirim et al. 2008). Studies have also demonstrated that DSPP, and Col I. We found that ALP protein activity and the spontaneous induction of osteoclast differentiation occurred in formation of mineralized nodules were significantly reduced cocultures of human DPCs and human CD14+ cells (Lossdorfer and that the ALP, OCN, and DSPP genes (mRNA) were downet al. 2009). regulated. Surprisingly, there was no effect on Col I expression, In this study, we examined the effect of RUNX2 mutation on but this is consistent with a previous study in RUNX2 homozy-/the ability of primary DPCs to support osteoclast differentiagous mutant (Runx2 ) mice (Aberg et al. 2004). Here, DSPP tion; we found reduced expression of the osteoclast-associated was found to be downregulated in odontoblasts, whereas the markers TRAP, CTSK, CTR, and MMP9 in the primary DPCs expression of Col I was not affected. Also, consistent with of the CCD patient, when cocultured with osteoclast precursor what we found, Ding et al. (2013) found that the proliferative cells (PBMCs). Another study also found that when osteoblasts capacity and osteogenic potential of MSCs isolated from the from the skull of Runx2-/- mice were cultured with normal bone marrow and dental pulp of a CCD patient were decreased spleen cells, the ability of osteoblasts to induce osteoclast difin comparison with those of normal individuals. These results ferentiation was significantly reduced (Thirunavukkarasu et al. indicate that RUNX2 mutation may reduce the ability of primary 2000). Therefore, CCD is a natural model for investigating the DPCs in patients with CCD to differentiate and mineralize, effect of RUNX2 mutation on bone remodeling. which may be a cause of dental abnormalities in these patients.
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Figure 3. RUNX2 mutation reduced the expression of some odontoblast-associated genes in primary dental pulp cells (DPCs) from cleidocranial dysplasia patient. DPCs from normal control (RUNX2+/+) and cleidocranial dysplasia patient (RUNX2+/m) were isolated and incubated in osteogenic induction medium for 7 d. (A–D) Quantitative reverse transcription polymerase chain reaction analysis RUNX2, OCN, DSPP, and Col I mRNA levels on day 7. *P < 0.05. **P < 0.01. (E) The expression of DSPP and RUNX2 was examined by Western blot in primary DPCs on day 7.
Figure 4. RUNX2 mutation reduced the expression of osteoclast-related genes and the boneremodeling activity. (A–D) Primary dental pulp cells (DPCs) were cocultured with peripheral blood mononuclear cells for 7 d; then, RNA was isolated, and quantitative reverse transcription polymerase chain reaction was used to analyze TRAP, CTSK, CTR, and MMP9 in mRNA levels. (E–F) Quantitative reverse transcription polymerase chain reaction analysis of RANKL and OPG mRNA levels in primary DPCs from cleidocranial dysplasia patient and normal control. (G) The ratio of RANKL/OPG in primary DPCs. *P < 0.05. **P < 0.01. ***P < 0.001.
The RANKL/RANK/OPG signaling pathway is considered a classical pathway regulating osteoclast formation. RANKL and macrophage colony-stimulating factor produced by osteoblasts/ stromal cells stimulates the differentiation and fusion of osteoclast progenitors to produce functional osteoclasts (Tanaka et al. 1993). OPG, a soluble decoy receptor for RANKL, inhibits the effects of RANKL (Troen 2003). The ratio of RANKL/OPG plays a key role in deciding the local balance between osteoblasts and osteoclasts (Hofbauer and Schoppet 2004). We found that the RANKL mRNA levels in the CCD primary DPCs were reduced while OPG was increased. Therefore, the RUNX2 mutation reduced the ratio of RANKL/OPG, indicating reduced osteoclastic activity and interference in the balance between
odontoblasts and odontoclasts that could impair the root resorption process. In support of this result, other studies have found that the ratio of RANKL/OPG in periodontal ligament cells and follicle cells of CCD patients was lower than normal (Lossdorfer et al. 2009; Dorotheou et al. 2013), and an animal study performed in Runx2-/- mice found that the expression of OPG was upregulated and that RANKL was downregulated in osteoblasts from the calvaria of Runx2-/- mice (Cogulu et al. 2004). Another study reported that RUNX2 regulated the expression of RANKL and OPG as well as the osteoclast-inducing capacity of periodontal ligament stem cells (Li et al. 2014). However, studies regarding local expression of RANKL and OPG in DPCs of CCD patients have not yet been reported. In summary, we found that the primary DPCs of a CCD patient exhibit reduced proliferation and mineralization and a reduced capacity to support osteoclast differentiation. We conclude that these effects were the result of the CCDassociated RUNX2 mutation, which was also found to interfere with odontoblast differentiation and the ability of primary DPCs to induce osteoclastic differentiation of precursor cells. These results shed significant light on the dental defects and primary tooth retention seen in CCD patients.
Author Contributions
W.J. Yan, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; S.G. Zheng, contributed to conception, design, data acquisition, analysis, and interpretation, critically revised the manuscript; C.Y. Zhang, contributed to design, drafted and critically revised the manuscript; Y.X. Wang, contributed to design, critically revised the manuscript; X. Yang, contributed to data acquisition, critically revised the manuscript; Z.N. Liu, contributed to data analysis and interpretation, critically revised the manuscript; X.Z. Wang, X.Y. Sun, contributed to data analysis, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81070815). We are grateful to the
Abnormal Differentiation of Dental Pulp Cells participant and his family members for their participation and contributions. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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583 Lossdorfer S, Abou Jamra B, Rath-Deschner B, Götz W, Abou Jamra R, Braumann B, Jäger A. 2009. The role of periodontal ligament cells in delayed tooth eruption in patients with cleidocranial dysostosis. J Orofac Orthop. 70:495–510. Mundlos S. 1999. Cleidocranial dysplasia: clinical and molecular genetics. J Med Genet. 36:177–182. Mundlos S, Otto F, Mundlos C, Mulliken JB, Aylsworth AS, Albright S, Lindhout D, Cole WG, Henn W, Knoll JH, et al. 1997. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell. 89:773–779. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, et al. 1997. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. 89:765–771. Quinn JM, Neale S, Fujikawa Y, McGee JO, Athanasou NA. 1998. Human osteoclast formation from blood monocytes, peritoneal macrophages, and bone marrow cells. Calcif Tissue Int. 62:527–531. Sahara N, Okafuji N, Toyoki A, Suzuki I, Deguchi T, Suzuki K. 1992. Odontoclastic resorption at the pulpal surface of coronal dentin prior to the shedding of human deciduous teeth. Arch Histol Cytol. 55:273–285. Sato S, Kimura A, Ozdemir J, Asou Y, Miyazaki M, Jinno T, Ae K, Liu X, Osaki M, Takeuchi Y, et al. 2008. The distinct role of the Runx proteins in chondrocyte differentiation and intervertebral disc degeneration: findings in murine models and in human disease. Arthritis Rheum. 58:2764–2775. Shaikh R, Shusterman S. 1998. Delayed dental maturation in cleidocranial dysplasia. ASDC J Dent Child. 65:325–329, 355. Suzuki T, Suda N, Ohyama K. 2004. Osteoclastogenesis during mouse tooth germ development is mediated by receptor activator of NFKappa-B ligand (RANKL). J Bone Miner Metab. 22:185–191. Tanaka S, Takahashi N, Udagawa N, Tamura T, Akatsu T, Stanley ER, Kurokawa T, Suda T. 1993. Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors. J Clin Invest. 91:257–263. Thirunavukkarasu K, Halladay DL, Miles RR, Yang X, Galvin RJ, Chandrasekhar S, Martin TJ, Onyia JE. 2000. The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function. J Biol Chem. 275:25163–25172. Troen BR. 2003. Molecular mechanisms underlying osteoclast formation and activation. Exp Gerontol. 38:605–614. Trowbridge HO. 2003. Pulp biology: progress during the past 25 years. Aust Endod J. 29:5–12. Tsukamoto Y, Fukutani S, Shin-Ike T, Kubota T, Sato S, Suzuki Y, Mori M. 1992. Mineralized nodule formation by cultures of human dental pulpderived fibroblasts. Arch Oral Biol. 37:1045–1055. Uchiyama M, Nakamichi Y, Nakamura M, Kinugawa S, Yamada H, Udagawa N, Miyazawa H. 2009. Dental pulp and periodontal ligament cells support osteoclastic differentiation. J Dent Res. 88:609–614. Xuan D, Sun X, Yan Y, Xie B, Xu P, Zhang J. 2010. Effect of cleidocranial dysplasia-related novel mutation of RUNX2 on characteristics of dental pulp cells and tooth development. J Cell Biochem. 111:1473–1481. Yildirim S, Yapar M, Sermet U, Sener K, Kubar A. 2008. The role of dental pulp cells in resorption of deciduous teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 105:113–120. Zhang C, Zheng S, Wang Y, Zhao Y, Zhu J, Ge L. 2010. Mutational analysis of RUNX2 gene in Chinese patients with cleidocranial dysplasia. Mutagenesis. 25:589–594.
research-article2015
JDRXXX10.1177/0022034514566655Journal of Dental ResearchAbnormal Differentiation of Dental Pulp Cells
Research Reports: Biological
Abnormal Differentiation of Dental Pulp Cells in Cleidocranial Dysplasia
Journal of Dental Research 2015, Vol. 94(4) 577–583 © International & American Associations for Dental Research 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0022034514566655 jdr.sagepub.com
W.J. Yan1,2*, C.Y. Zhang1*, X. Yang3, Z.N. Liu4, X.Z. Wang1, X.Y. Sun1, Y.X. Wang5, and S.G. Zheng1
Abstract Cleidocranial dysplasia (CCD) is a skeletal dysplasia caused by heterozygous mutations of RUNX2, a gene that is essential for the mineralization of bone and tooth. We isolated primary dental pulp cells from a 10-y-old patient and tested their proliferative capacity, alkaline phosphatase activity, and ability to form mineralized nodules, in comparison with those from 7 healthy children. All these measures were reduced in primary dental pulp cells from the CCD patient. The expression of the osteoblast/odontoblast-associated genes RUNX2, ALP, OCN, and DSPP was also found to be significantly decreased in the primary dental pulp cells of the CCD patient. The osteoclast-related markers TRAP, CTSK, CTR, and MMP9 were decreased in primary dental pulp cells cocultured with human peripheral blood mononuclear cells. Moreover, the expression of RANKL and the ratio of RANKL/OPG were both reduced in the cells from the CCD patient, indicating that the RUNX2 mutation interfered with the bone-remodeling pathway and decreased the capacity of primary dental pulp cells to support osteoclast differentiation. These effects may be partly responsible for the defects in tooth development and the retention of primary teeth that is typical of CCD. Keywords: cell biology, craniofacial anomalies, bone remodeling/regeneration, craniofacial biology/genetics, osteogenesis, osteoclasts
Introduction Cleidocranial dysplasia (CCD; MIM 119600) is an inherited autosomal-dominant bone disease that is characterized by hypoplasia of the clavicle and dental abnormalities (Cooper et al. 2001). The main systemic manifestations include delayed closure of fontanelle or persistently open fontanelle, widened suture and the sutural bone (Wormian bones), unilateral/bilateral clavicle hypoplasia or absence, short stature, and other skeletal anomalies (Mundlos 1999). Dental abnormalities mainly consist of primary teeth retention, delayed eruption of permanent dentition, and supernumerary teeth (Jensen and Kreiborg 1991; Shaikh and Shusterman 1998). It has been demonstrated that heterozygous mutation of the RUNX2 gene (synonyms: CBFA1, AML3, OSF2, PEBP2A) is responsible for CCD (Mundlos et al. 1997; Otto et al. 1997). This osteoblast-specific transcription factor, which is mapped to human chromosome 6p21, is involved in regulation of bone metabolism and plays an important role in osteoblast differentiation and osteoclast formation. It has been shown that RUNX2 specifically regulates the transcription and expression of some genes related to bone and tooth development, including osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (Col I), osteopontin (OPN), bone sialoprotein (BSP), and ameloblastin (AMBN; Ducy et al. 1997). Runx2 homozygous mutant mice exhibit lack of bone formation owing to maturational arrest of osteoblasts (Komori et al. 1997), while Runx2 heterozygous mutant mice exhibit specific characteristics of CCD, such as hypoplastic nasal bones and clavicles (Otto et al.
1997). RUNX2 also plays an important role in the differentiation of osteoclasts and odontoclasts through its regulation of the RANKL/RANK/OPG signaling pathway (Sato et al. 2008). RUNX2 binding elements have been found in the promoter region of OPG and RANKL (Aberg et al. 2004), indicating that the expression of OPG and RANKL could be directly regulated by RUNX2. 1
Department of Preventive Dentistry, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China 2 Department of Pediatric Dentistry, the First Division, Peking University School and Hospital of Stomatology, Xicheng District, Beijing, China 3 Department of Stomatology, Dongzhimen Hospital Beijing University of Chinese Medicine, Dongcheng District, Beijing, China 4 Department of Prosthodontics, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China 5 Central Laboratory, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China *First coauthors contributing equally to this article. A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. Corresponding Authors: S.G. Zheng, Department of Preventive Dentistry, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, China. Email: [email protected] Y.X. Wang, Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidan District, Beijing 100081, China. Email: [email protected]
578 The mechanism producing dental abnormalities in CCD patients is not fully understood. However, it is well known that primary dental pulp cells (DPCs) play an important role in the development and eruption of permanent successors (Yildirim et al. 2008); therefore, changes to the biological characteristics of primary DPCs could lead to some of the dental abnormalities associated with CCD. In this study, primary DPCs of a patient with CCD (a natural RUNX2 gene mutation model) were examined to see if their proliferation, mineralization, or differentiation behavior differed from those of healthy controls.
Materials and Methods Patient This study was approved by the Ethical Committee of Peking University School of Stomatology (approval No. PKUS SIRB2012004). A Chinese child aged 10 y old who was clinically and genetically diagnosed with CCD was examined with informed parental consent (Zhang et al. 2010).
Cell Culture Retained deciduous mandibular lateral incisors of the CCD patient were extracted; one third of the root length had been resorbed. Briefly, dental pulp was isolated, and 2 mm from the apical region was discarded; then, the remainder was digested in a mixture of collagenase type I (3 mg/mL; Sigma-Aldrich, MO, USA) and dispase (4 mg/mL; Sigma-Aldrich), and the single-cell suspensions were incubated in DMEM medium (Gibco, MO, USA) containing 10% fetal bovine serum (Hyclone, UT, USA). Cells were incubated at 37 °C in 5% CO2. The third to fifth passage of the primary DPCs was used for the experiments. Nine retained mandibular deciduous incisors from 7 unaffected children aged 6 to 8 y old were used as controls. The extracted incisors had a similar pattern of root resorption to those of the CCD patient.
Immunohistochemical Staining for Cell Identification Primary DPCs were plated on coverslips at 2 × 104 cells per well in a 24-well plate. When 70% confluency was reached, the cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 min and then permeabilized with 0.2% Triton X-100 in phosphate buffered saline for 10 min. Next, the cells were incubated overnight at 4 °C with primary antibodies against cytokeratin and vimentin (Zhongshan Bioengineering Co. Ltd., China). Then the procedure was performed according to the protocol of an SP immunohistochemical kit and a 3,3′-diaminobenzidine coloration kit (Zhongshan Bioengineering Co. Ltd.). Adipocytes and squamous carcinoma cells were used as positive controls for vimentin and keratin staining. The results were observed with a light microscope equipped with a camera (BX51 microscope and DP72 camera; Olympus, Japan).
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Cell Proliferation Assay Primary DPCs were plated in 96-well plates (2 × 103 cells per well) containing DMEM growth (basal) medium. After 12 h, a cell proliferation assay was performed with a Cell Counting Kit 8 (Sigma) according to the manufacturer’s instructions. Absorbance was measured at 450 nm with an ELx808 absorbance microplate reader (BioTeK, USA).
ALP and Alizarin Red Staining Primary DPCs were plated in 12-well plates at a density of 2 × 104 cells per well. At 80% confluency, the growth medium was replaced with osteogenic medium, composed of 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 10 nM L-ascorbic acid in growth medium. After 7 d, ALP staining was performed with an ALP histochemical staining kit (Cwbiotech, Beijing, China), and ALP activity was analyzed with an ALP activity assay kit (Jiancheng, Nanjing, China) according to the manufacturer’s instructions. Primary DPCs were seeded into 12-well plates (2 × 104 cells per well). After 24 h, they were treated with osteogenic medium and cultured for another 21 d. Then, they were fixed with 4% paraformaldehyde and stained with alizarin red solution (Sigma-Aldrich).
Quantitative Reverse Transcription Polymerase Chain Reaction Primary DPCs were seeded into 6-well plates (1.2 × 105 cells per well). At 80% confluency, the growth medium was replaced with osteogenic medium. After 7 d, the total RNA was extracted with Trizol (Invitrogen, CA, USA), and the RNA was reverse transcribed as previously described (Liu et al. 2013). The resultant cDNAs were amplified through the specific sets of primers for ALP, DSPP, OCN, and Col I. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used to normalize RNA expression levels. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed with a SYBR Green PCR kit (Roche Applied Science, IN, USA) according to the manufacturer’s protocol, and the results were assessed with 7500 Software 2.0.1 (Applied Biosystems, CA, USA).
Western-Blot Analysis Primary DPCs (4 × 105 cells) were seeded on 100-mm dishes. When 80% confluency was reached, the growth medium was replaced with osteogenic medium, and they were incubated in this for 7 d. Then the total protein was extracted with a protein lysis buffer (Applygen, Beijing, China). Protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal aliquots of total protein (40 μg) were electrophoresed in a 7.5% SDS-PAGE gel and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were incubated in 5% nonfat dry
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Abnormal Differentiation of Dental Pulp Cells milk for 1 h to block the nonspecific binding sites and incubated with primary antibodies against DSPP, RUNX2, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated anti-rabbit or anti-mouse IgG (Jackson Immuno Research, PA, USA) were then used, and immunoreactive bands were detected using a western enhanced chemiluminescence blotting kit (Applygen Technology Inc., Beijing, China).
Coculture of Primary DPCs and Human Peripheral Blood Mononuclear Cells Human peripheral blood from 2 healthy volunteers was layered on a Ficoll-Paque density gradient and centrifuged at 1800 rpm for 30 min. The white membrane layer between the FicollPaque and blood plasma was collected; this mainly consists of peripheral blood mononuclear cells (PBMCs). The primary DPCs were plated in 24-well plates (3 × 104 cells per well). When 60% confluency was reached, PBMCs were plated into the same wells at 3 × 106 cells per well. After 7 d, the mRNA of the cells was extracted, and real-time polymerase chain reaction was performed with a SYBR Green PCR kit to investigate the expression of the osteoclast-associated genes TRAP, CTSK, CTR, and MMP9. Then, the ratio of RANKL/OPG was measured for the primary DPCs.
Statistical Analysis Data are expressed as means ± SD. Differences between groups were determined by 1-way analysis of variance. The threshold for statistical significance was set at P < 0.05.
Results Clinical Manifestation of CCD in the Patient This patient is a family case whose father was also affected by CCD. The patient showed typical CCD features, including hypoplastic clavicles, dental abnormalities, and classic craniofacial features, such as insufficiently developed midface and prominent forehead (Appendix Fig.). Dental anomalies included retained deciduous teeth, delayed eruption of permanent teeth, and supernumerary teeth (Appendix Fig. C–F). Although this patient was already 10 y old, all the teeth in the mouth were deciduous except for the first permanent molars and the lower left incisor. In a previous study, we used mutation analysis to identify a heterozygous frame shift mutation in the RUNX2 gene of this patient (c.514delT, p.Ser172fs; Zhang et al. 2010); this confirmed the clinical diagnosis of CCD.
RUNX2 Mutation Decreased the Growth Rate of Primary DPCs Immunohistochemical staining was first performed to verify the source of the primary DPCs. The DPCs from the CCD
patient and normal control were positive for vimentin staining and negative for keratin staining, indicating that the isolated cells were mesenchymal cells (Fig. 1A). The morphology of the primary DPCs was spindle shaped, similar to fibroblasts for CCD (RUNX2+/m) and normal cells (RUNX2+/+; Fig. 1B). However, the growth rate was observed to be slower for CCD versus normal cells, becoming noticeable on day 3 (Fig. 1B). Quantitative analysis through a Cell Counting Kit 8 proliferation assay confirmed this decrease, with significantly lower cell numbers being found for days 2 to 10 (Fig. 1C).
RUNX2 Mutation Reduced the Mineralization Capacity of Primary DPCs ALP production, which is regulated by RUNX2, is one of the main indicators of mineralization and odontoblast differentiation (Trowbridge 2003). Enhanced ALP activity is considered an early marker of DPC differentiation and dentin formation (Tsukamoto et al. 1992). Therefore, the effect of the CCDassociated RUNX2 mutation on ALP expression was assessed. The basal level of ALP mRNA in the CCD patient’s DPCs was reduced by 79% compared with the normal control (Fig. 2A). A similar result was found after stimulation with osteogenic medium; 63% reduction versus the control (Fig. 2A). ALP staining was performed to investigate the effect of RUNX2 gene mutation on ALP expression at the protein level. ALP staining showed a lower ALP expression in the CCD patient (Fig. 2B). Quantitative analysis showed that ALP protein levels were reduced by approximately 30% in the CCD cells, under both basal and osteogenic conditions (Fig. 2C); this indicates reduced mineralization. Alizarin red staining was then performed to study the effect of RUNX2 mutation on the formation of mineralized nodules. In agreement with the ALP results, the number of mineralized nodules in the CCD cell cultures was noticeably reduced when compared with control cultures, after stimulation in osteogenic medium (Fig. 2D).
RUNX2 Mutation Reduced the Expression of Osteoblast/Odontoblast-associated Genes in Primary DPCs The expression of osteoblast/odontoblast-associated genes in the primary DPCs was analyzed. DSPP, OCN, and Col I were chosen for analysis because they are potential downstream targets of RUNX2 (Ducy et al. 1997; Chen et al. 2002). For primary DPCs from the CCD patient, RUNX2 mRNA levels were reduced about 60% in both basal and osteogenic media (Fig. 3A), and the mRNA levels of OCN and DSPP showed a similar pattern (Fig. 3B, C), whereas Col I levels remained unchanged (Fig. 3D). RUNX2 and DSPP protein levels were detected by Western blot. In agreement with the mRNA measurements, the expression of RUNX2 and DSPP protein was reduced in the CCD cells in both media (Fig. 3E).
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Figure 1. The proliferation of primary dental pulp cells (DPCs) was delayed by RUNX2 mutation. (A) Vimentin and keratin immunohistochemical analyses for primary DPCs from normal control and cleidocranial dysplasia (CCD) patient. ADPs, adipocytes; RUNX2+/+, normal control; RUNX2+/m, CCD patient; SCCs, squamous carcinoma cells. (B) Representative photomicrographs of primary DPCs from normal control and CCD patient at indicated time point after incubation. (C) Proliferation assay for primary DPCs from normal control and CCD patient at indicated time point after incubation. *P < 0.05. **P < 0.01.
RUNX2 Mutation Reduced the Expression of Osteoclast-related Genes and Modified Bone Remodeling Activity Osteoblasts and other mesenchyme-derived cells can induce differentiation of osteoclast precursors into osteoclasts (Fujikawa et al. 1996; Quinn et al. 1998), and DPCs have been shown to induce differentiation of PBMCs into osteoclasts (Uchiyama et al. 2009). Through a coculture composed of PBMCs and primary DPCs, the expression of osteoclastrelated genes was measured to assess the effect of CCD and normal primary DPCs on the differentiation of PBMCs into osteoclasts. The mRNA levels of TRAP, CTSK, CTR, and MMP9 were all reduced by the RUNX2 mutation in the primary DPCs from the CCD patient (Fig. 4A–D), indicating decreased osteoclast activity in the coculture. The RANKL/OPG signaling pathway plays an important role in the differentiation and maturation process of osteoclasts
(Suzuki et al. 2004). RANKL mRNA levels were reduced by 90% in the CCD case compared with the control (Fig. 4E). In addition, a 35% increase in OPG mRNA was found for the CCD DPCs (Fig. 4F). Accordingly, the ratio of RANKL/OPG was reduced by about 92% (Fig. 4G), indicating impairment of the RANKL/OPG signaling pathway when the function of RUNX2 is disturbed by heterozygous mutation.
Discussion Although there are numerous skeletal deficiencies in CCD patients, the dental disorder is most often their primary concern and major cause of reduced quality of life. The patient included in this study had typical features of CCD, such as hypoplastic clavicles and dental abnormalities. A heterozygous mutation in RUNX2 was detected in this patient, further confirming the causal role of RUNX2 mutation in CCD (Mundlos et al. 1997). To explain the typical dental anomalies of this patient, we
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hypothesized that the CCDassociated RUNX2 mutation may affect the mineralization capacity of the patient’s primary DPCs along with their ability to induce osteoclast differentiation. We found that ALP activity and mineralization of the primary DPCs were reduced by RUNX2 mutation. The expression of osteoblast/ odontoblast-related genes, including RUNX2, ALP, OCN, and DSPP, was significantly decreased in primary DPCs from the CCD patient. So, mineralization capacity of primary DPCs was reduced by the RUNX2 mutation. We found that the growth rate of primary DPCs from the CCD patient was slower than normal control. Figure 2. RUNX2 mutation reduced the alkaline phosphatase (ALP) activity and mineralization of This is consistent with a previous primary dental pulp cells (DPCs). DPCs from normal control (RUNX2+/+) and cleidocranial dysplasia study on DPCs (Xuan et al. 2010). (CCD) patient (RUNX2+/m) were isolated and cultured in osteogenic medium, which was composed of RUNX2 plays an important role in 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 10 nM L-ascorbic acid for 7 or 21 d. proliferation of chondrocytes (Hinoi (A) The ALP mRNA levels of normal and CCD patient DPCs were detected by quantitative reverse transcription polymerase chain reaction after incubating in osteogenic medium for 7 d. (B) The ALP et al. 2006); it is also involved in staining of DPCs from normal control and CCD patient was tested by ALP histochemical staining on regulating the expression of cell day 7. (C) ALP activity for DPCs from normal control and CCD patient was quantified on day 7. **P cycle–related genes, such as TGFB< 0.01. (D) Typical mineralized nodules stained with alizarin red for DPCs from normal control and CCD patient on day 21. RII, VEGFB, and BMP2 (Ji et al. 2001; Chen et al. 2005). However, Retention of the primary teeth and delayed eruption of perthe role of RUNX2 in proliferation of primary DPCs needs to be manent dentition are the most common oral manifestations in further investigated. CCD patients. The exfoliation of deciduous teeth is a complex As a transcription factor, RUNX2 plays an essential role in and orderly physiologic process, which involves a balance the development of bone and tooth (Ducy et al. 1997; D’Souza between osteoblast/odontoblast and osteoclast/odontoclast et al. 1999). Many related genes are regulated by RUNX2, activity. Periodontal ligament cells and dental follicle cells play such as OCN, ALP, Col I, OPN, BSP, and AMBN (Ducy et al. an important role in the process, and primary DPCs have also 1997). Among them, ALP and OCN are respectively considbeen shown to be involved. The odontoclastic resorption of ered early- and late-stage markers of DPC differentiation and coronal dentin takes place just before shedding (Sahara et al. dentin formation. DSPP is recognized as a specific marker of 1992). Primary DPCs can produce cytokines that induce cells of odontoblast differentiation, and there are RUNX2 binding sites the monocyte-macrophage lineage to become osteoclasts/odonin the regulatory elements of the mouse Dspp gene (Chen et al. toclasts. During physiologic resorption, RANKL and CSF-1 2002). Therefore, this study explored the effect of the CCDmRNA expression was shown to increase in primary DPCs associated RUNX2 mutation on the expression of ALP, OCN, (Yildirim et al. 2008). Studies have also demonstrated that DSPP, and Col I. We found that ALP protein activity and the spontaneous induction of osteoclast differentiation occurred in formation of mineralized nodules were significantly reduced cocultures of human DPCs and human CD14+ cells (Lossdorfer and that the ALP, OCN, and DSPP genes (mRNA) were downet al. 2009). regulated. Surprisingly, there was no effect on Col I expression, In this study, we examined the effect of RUNX2 mutation on but this is consistent with a previous study in RUNX2 homozy-/the ability of primary DPCs to support osteoclast differentiagous mutant (Runx2 ) mice (Aberg et al. 2004). Here, DSPP tion; we found reduced expression of the osteoclast-associated was found to be downregulated in odontoblasts, whereas the markers TRAP, CTSK, CTR, and MMP9 in the primary DPCs expression of Col I was not affected. Also, consistent with of the CCD patient, when cocultured with osteoclast precursor what we found, Ding et al. (2013) found that the proliferative cells (PBMCs). Another study also found that when osteoblasts capacity and osteogenic potential of MSCs isolated from the from the skull of Runx2-/- mice were cultured with normal bone marrow and dental pulp of a CCD patient were decreased spleen cells, the ability of osteoblasts to induce osteoclast difin comparison with those of normal individuals. These results ferentiation was significantly reduced (Thirunavukkarasu et al. indicate that RUNX2 mutation may reduce the ability of primary 2000). Therefore, CCD is a natural model for investigating the DPCs in patients with CCD to differentiate and mineralize, effect of RUNX2 mutation on bone remodeling. which may be a cause of dental abnormalities in these patients.
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Figure 3. RUNX2 mutation reduced the expression of some odontoblast-associated genes in primary dental pulp cells (DPCs) from cleidocranial dysplasia patient. DPCs from normal control (RUNX2+/+) and cleidocranial dysplasia patient (RUNX2+/m) were isolated and incubated in osteogenic induction medium for 7 d. (A–D) Quantitative reverse transcription polymerase chain reaction analysis RUNX2, OCN, DSPP, and Col I mRNA levels on day 7. *P < 0.05. **P < 0.01. (E) The expression of DSPP and RUNX2 was examined by Western blot in primary DPCs on day 7.
Figure 4. RUNX2 mutation reduced the expression of osteoclast-related genes and the boneremodeling activity. (A–D) Primary dental pulp cells (DPCs) were cocultured with peripheral blood mononuclear cells for 7 d; then, RNA was isolated, and quantitative reverse transcription polymerase chain reaction was used to analyze TRAP, CTSK, CTR, and MMP9 in mRNA levels. (E–F) Quantitative reverse transcription polymerase chain reaction analysis of RANKL and OPG mRNA levels in primary DPCs from cleidocranial dysplasia patient and normal control. (G) The ratio of RANKL/OPG in primary DPCs. *P < 0.05. **P < 0.01. ***P < 0.001.
The RANKL/RANK/OPG signaling pathway is considered a classical pathway regulating osteoclast formation. RANKL and macrophage colony-stimulating factor produced by osteoblasts/ stromal cells stimulates the differentiation and fusion of osteoclast progenitors to produce functional osteoclasts (Tanaka et al. 1993). OPG, a soluble decoy receptor for RANKL, inhibits the effects of RANKL (Troen 2003). The ratio of RANKL/OPG plays a key role in deciding the local balance between osteoblasts and osteoclasts (Hofbauer and Schoppet 2004). We found that the RANKL mRNA levels in the CCD primary DPCs were reduced while OPG was increased. Therefore, the RUNX2 mutation reduced the ratio of RANKL/OPG, indicating reduced osteoclastic activity and interference in the balance between
odontoblasts and odontoclasts that could impair the root resorption process. In support of this result, other studies have found that the ratio of RANKL/OPG in periodontal ligament cells and follicle cells of CCD patients was lower than normal (Lossdorfer et al. 2009; Dorotheou et al. 2013), and an animal study performed in Runx2-/- mice found that the expression of OPG was upregulated and that RANKL was downregulated in osteoblasts from the calvaria of Runx2-/- mice (Cogulu et al. 2004). Another study reported that RUNX2 regulated the expression of RANKL and OPG as well as the osteoclast-inducing capacity of periodontal ligament stem cells (Li et al. 2014). However, studies regarding local expression of RANKL and OPG in DPCs of CCD patients have not yet been reported. In summary, we found that the primary DPCs of a CCD patient exhibit reduced proliferation and mineralization and a reduced capacity to support osteoclast differentiation. We conclude that these effects were the result of the CCDassociated RUNX2 mutation, which was also found to interfere with odontoblast differentiation and the ability of primary DPCs to induce osteoclastic differentiation of precursor cells. These results shed significant light on the dental defects and primary tooth retention seen in CCD patients.
Author Contributions
W.J. Yan, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; S.G. Zheng, contributed to conception, design, data acquisition, analysis, and interpretation, critically revised the manuscript; C.Y. Zhang, contributed to design, drafted and critically revised the manuscript; Y.X. Wang, contributed to design, critically revised the manuscript; X. Yang, contributed to data acquisition, critically revised the manuscript; Z.N. Liu, contributed to data analysis and interpretation, critically revised the manuscript; X.Z. Wang, X.Y. Sun, contributed to data analysis, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81070815). We are grateful to the
Abnormal Differentiation of Dental Pulp Cells participant and his family members for their participation and contributions. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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