Transcription Factors for Dental Stem Cell Differentiation

Sandra Viale-Bouroncle, MSc1/Oliver Felthaus, Dipl Biol1/Gottfried Schmalz, DDS, Dr med dent, PhD2/ Torsten E. Reichert, DDS, DMD, PhD, MD, Dr med, Dr med dent2/ Christian Morsczeck, Dipl Biol, Dr rer nat (PhD)3

Dental stem cells are excellent for oral and craniofacial tissue engineering. A profound knowledge about molecular processes in dental stem cells is necessary to create treatment approaches in oral medicine. Transcription factors regulate gene expression and provide decisive information for cellular functions. In recent years, the authors have investigated transcriptomes in dental stem cells before and after osteogenic differentiation. The present paper reports on the potential role of selected transcription factors, including ZBTB16, TP53, and SP1, in dental stem cell differentiation. This review discusses putative molecular processes in dental stem cells and summarizes the current knowledge. Oral CraniOfaC Tissue eng 2011;1:306314
Key words: dental stem cells, differentiation, molecular biology, transcription factors

omatic stem cells such as bone marrow mesenchymal stem cells (BMMSCs) are multipotent and can give rise to a variety of differentiated lineages, including osteogenic, chondrogenic, and adipogenic cells. This offers the opportunity for the development of stem cellbased therapies. Since BMMSCs are rare and difficult to obtain, it is desirable to find alternative sources for multipotent somatic stem cells. Among many other tissues, somatic stem cells have been found in dental tissues. To date, at least five different types of dental stem cells have been isolated1: postnatal dental pulp stem cells (DPSCs),2 stem cells from exfoliated deciduous

1PhD

students, Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany. 2Director and Professor, Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany. 3Group Leader and Lecturer, Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany. Correspondence to: Dr Christian Morsczeck, Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany. Email: christian.morsczeck@klinik. uni-regensburg.de Sandra Viale-Bouroncle and Oliver Felthaus contributed equally to this work.

(primary) teeth (SHED),3 periodontal ligament stem cells (PDLSCs),4 precursor cells from the dental follicle (dental follicle cells [DFCs]; Fig 1),5 and stem cells from apical papilla (SCAP).6 Isolation of another dental cell population, dental neural crestderived progenitor cells (dNC-PCs), was reported recently.7 These cells are derived from the apical pad of an unerupted impacted tooth germ. All aforementioned stem cells from dental tissues share characteristics of BMMSCs, such as clonogenicity and plastic adherence. Most of these cell populations have a Stro1positive subpopulation and can be induced in vitro to express osteogenic marker genes. On the other hand, DPSCs, SHED, SCAP, PDLSCs, DFPCs, and dNC-PCs share some characteristics that distinguish them from BMMSCs. During development, cells that eventually form the dental mesenchyme derive from the neural crest. Therefore, tooth mesenchyme is referred to as ectomesenchyme. This might be one reason that dental stem cells are in a less-differentiated/ lineage-restricted state. Undifferentiated cells with an ectomesenchymal origin have a better neural differentiation potential than mesenchymal stem cells such as BMMSCs.8 Additionally, dental stem cells should possess a better odontogenic differentiation potential than BMMSCs. DPSCs and SHED in particular can be induced to express dentin sialophosphoprotein and dentin matrix acidic phosphoprotein1,3,9,10 which are considered odontogenic marker genes.11 All these features make dental stem cells an important source

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Fig 1 Cell morphology of DFCs was visualized by staining of actin filaments with Rhodamine phalloidin and the cell nucleus with Hoechst 33258 after 24 hours in cell culture. Bar = 20 m.

for regenerative dentistry, but little is known about the molecular processes involved in proliferation and differentiation of these cells. Transcription factors (TFs) drive these processes in cells and are therefore important targets for studies of dental stem cells.

TranSCripTion FaCTorS
The transcription of genes is facilitated by the activity of the RNA polymerase. The activity of the RNA polymerases is complex and highly regulated via a number of further proteins, which are needed for the initiation of transcription. Among other special types of RNA polymerases in plants, mitochondria, and chloroplasts, there are three general types of RNA polymerases in eukaryotes: RNA polymerase (RNAP) I, RNAP II, and RNAP III. RNAP I synthesizes a 45S preribosomal RNA (rRNA), which is subsequently processed into the ribosomal 28S, 18S, and 5.8S rRNAs.12 RNAP III is responsible for the transcription of the transfer RNAs (tRNAs) and the 5S rRNA.13 Normally, the requirements for rRNAs and tRNAs do not vary greatly between different cell types and conditions. Synthesis of messenger RNA (mRNA), on the other hand, needs to be highly regulated. Every cell type expresses a different set of proteins, and the cell reacts to changing conditions with a changed gene expression profile. The synthesis of the protein-coding mRNA (as well as synthesis of small

nuclear RNA and microRNA) is accomplished by RNAP II.14 Expression of mRNA requires a high level of regulation; therefore, several DNA-binding proteins are needed for RNAP II to bind to the promoter and initiate transcription. These DNA-binding proteins are called TFs and can be subdivided into the general TFs and the gene regulatory proteins (specific TFs). General TFs are mandatory for transcription, are ubiquitously expressed in all cells, and do not play a role in the regulation of gene expression. Therefore they will not be discussed further in this article. Gene regulatory proteins, on the other hand, are expressed in specific tissues and regulate the expression pattern of the different cell types. They bind directly to specific DNA sequences (response element/TF-binding site) and can enhance (activators) or inhibit (repressors) the expression of a gene. Other proteins involved in transcription regulation, such as chromatin remodelers, histone acetylases, deacetylases, kinases, methylases, coactivators, and corepressors, do not bind to DNA directly.15 A gene regulatory protein typically contains three domains: a DNA-binding domain (DBD), with which the TF binds to specific promoter or enhancer sequences (response elements); a transactivating domain, to which coregulating proteins can bind; and a signal sensing domain, with which the TF can be activated as a response to external stimuli.16 The abundance of specific TFs (approximately 10% of all genes code for TFs) can be classified by the motif with which the protein binds to DNA.
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DiFFerenTiaTion
A prerequisite for differentiation is the existence of undifferentiated cells. In vivo, several factors are necessary for a cell to maintain its undifferentiated state. Among others, growth factors, cytokines, extracellular matrix, and cell-cell contacts (to other stem cells and to differentiated cells) are important for stem cells to keep their undifferentiated state. Taken together, these factors define the so-called stem cell niche,17,18 a microenvironment that sustains a stem cells undifferentiated condition. The stem cell niche is more than a mere location; it is a highly dynamic functional interplay between stem cells and their surrounding cells, structures, and molecules. Nevertheless, stem cells reside at specific sites in tissues. Staining with antibodies against Stro-1 and CD146 was done to identify the site of the dental stem cell niche. In dental pulp of primary and permanent teeth, Stro-1 and CD146positive cells are found in the perivascular region.19 Differentiation is a process that is accompanied by pronounced changes in the gene expression profile. These changes arise from the tissue-specific and highly regulated activity of specific TF/gene regulatory proteins. Specific TFs are activated by signaling pathways/growth factors and regulate the fate of the somatic stem cells. Many TFs are well known for their roles in osteogenic differentiation of mesenchymal stem cells of the bone marrow. Among these, DLX3, Dlx5, MSX-2, Runx2, and SP7 are the most important. They have been shown to regulate the expression of the osteogenic marker gene osteocalcin (OCN).18 Whereas Runx2 and Dlx5 promote osteogenic differentiation and OCN expression, MSX-2 inhibits osteogenic differentiation.18 In the periodontium, the activity of MSX-2 promotes commitment into periodontal ligament fibroblasts.19 Interestingly, during dexamethasone-induced osteogenic differentiation of DFCs, the up-regulation of these TFs was not as pronounced as expected.20 Whereas the up-regulation of mineralization-associated TFs was more pronounced after bone morphogenetic protein-2 (BMP-2)induced osteogenic differentiation, the dexamethasone-treated DFCs showed stronger biomineralization under in vitro conditions.20 Moreover, this improved differentiation was not inhibited after supplementation of cell culture conditions with the BMP antagonist Noggin. Therefore, it was hypothesized that there might be a BMP-2independent signaling network that improves osteogenic differentiation of the ectomesenchymally derived DFCs.20 Multiple promising candidates have emerged from previous studies. The strongest up-regulation in TFs was observed for the TFs ZBTB16 and NR4A3.20 During osteogenic differentiation in DFCs, 35% of all regulated genes have promoter binding 308
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sites for the TFs TP53 and SP1.21 DLX3, which is strongly up-regulated during BMP-2induced osteogenic differentiation of BMMSCs, is also up-regulated after dexamethasone-induced osteogenic differentiation of DFCs,17 but the response is even stronger after BMP-2induced differentiation. All these TFs might play important roles during mineralization of ectomesenchymally derived dental stem cells. Of special interest might be DLX3, which is up-regulated after the induction of differentiation with BMP-2 and dexamethasone. Therefore, DLX3 might be the key regulator of osteogenic differentiation in DFCs.

DLX3 anD CLaSSiCaL paThwayS oF oSTeogeniC DiFFerenTiaTion

The TF DLX3 (31.6 kDa) is a member of the DLX family and contains a homeodomain, which is related to the Distal-less domain of Drosophila. DLX3 is an essential TF for embryonic development,22 plays a decisive role in bone patterning and development, and can be detected in craniofacial bone.23,24 DLX3 is also expressed in otic and olfactory placodes, epidermis, the limb bud, branchial arches, the placenta, tooth germs, hair follicles, and osteoblasts.10 Moreover, analysis of normal and mutant DLX3 revealed that the influence of DLX3 expression on mineralized tissue development varies specifically according to the terminal differentiation for each cell type.2426 For example, a 4-bp mutation in DLX3 is related to the trichodentosseous syndrome, which is characterized by increased bone volume and mineral density and decreased thickness of dentin.2730 Recently, DLX3 was characterized as an important TF for the differentiation of DFCs into mineralized tissue cells such as alveolar bone osteoblasts (authors unpublished data). In comparison to BMMSCs, quantitative real-time polymerase chain reaction analysis revealed a different expression pattern of osteogenic markers in DFCs.17,18,24 Osteogenic TFs such as MSX2, RUNX2, DLX5, or OSTERIX were not regulated in DFCs during osteogenic differentiation. However, like BMMSCs, DLX3 expression was increased during osteogenic differentiation of DFCs.17 Differences in the expression patterns of osteogenic markers in MSCs and DFCs indicate that DLX3 differentially regulates bone development in both cell types. In osteoblast precursors, DLX3, together with DLX5, plays a major role in the transcriptional activity of OCN and in the induction of BMP-2mediated RUNX2 expression.18,31 DLX3 regulates directly the expression of the early and middle osteogenic markers alkaline phosphatase (ALP) and RUNX2, as well as ZBTB16, while it down-regulates the expression of

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the late osteogenic marker bone sialoprotein in DFCs (authors unpublished data). Furthermore, chromatin immunoprecipitation (ChIP) assays for protein-DNA interactions revealed that DLX3 binds directly to the RUNX2 promoter and regulates its expression (authors unpublished data) (Fig 2). DLX3 influence directly the matrix mineralization of differentiated DFCs. However, ALP activity and mineralization were observed only after induction with osteogenic differentiation medium (authors unpublished data). The present authors conducted a microarray study after DLX3 overexpression (microarray raw data accession number: GSE29753). The strongest upregulated gene after DLX3 overexpression was IL8. IL8 is expressed in human osteoblastlike cells such as osteosarcoma cell line MG-63, bone marrow stromal cells, and osteoclasts, suggesting that it plays a role in the regulation of cellular functions in bone.32,33 The chemokine interleukin-8 participates in most of the regulated pathways after DLX3 overexpression, such as the cytokine-cytokine receptor interaction pathway. However, preliminary experiments showed that interleukin-8 does not modulate the osteogenic differentiation of DFCs (authors unpublished data). Moreover, genes that are associated with developmental processes, such as BMP-2, NR4A2, HES1, and ATF3, were significantly up-regulated. The nuclear orphan transcription factor NR4A2 activates, for example, the promoter of osteopontin in osteoblasts34 and is another candidate for the differentiation of dental cells. In contrast, connective tissue markers such as type IIIA1 collagen (col3A1), elastin, osteomodulin, and PLXNC1 (plexin) were significantly down-regulated. After DLX3 overexpression, the Jak-STAT pathway, including genes sprouty2 (SPRY2) and sprouty4 (SPRY4), is highly up-regulated. SPRY2 and SPRY4 play relevant roles in the bidirectional signal among epithelial and mesenchymal cells, which is also important process for dental development.35,36 Furthermore, the Toll-like receptor (TLR) and Nod-like receptor (NLR) signaling pathways are also overrepresented and are well known for triggering the innate immune response. Although these pathways are associated with the immune response, new studies showed that both NLRs and TLRs also play a role in the regulation of differentiation in BMMSCs.36 Interestingly, within the most overrepresented network of associated functions after DLX3 overexpression in DFCs, terms appear such as inhibition of apoptosis, proliferation of normal cells, morphology of cells, chemotaxis of bone cell lines or binding of osteoblasts, and mobilization of calcium. Apoptosis, a fundamental component of osteoblast differentiation, contributes to maintenance of tissue organization and regulates bone homeostasis by bal-

BMP2

DLX3 IL-8? Proliferation Morphology Cell viability ALP Osteogenic differentiation

BSP ZBTB16

RUNX2 Osterix OCN

Fig 2 Schematic illustration of proposed regulation pathways and functions regulated by the TF DLX3 during osteogenic differentiation of osteoblasts/osteoblast progenitor cells. IL-8 = interleukin 8; BSP = bone sialoprotein.

ancing cell proliferation and cell death.3739 Furthermore, the influence of a DLX3 mutant for odontoblast apoptosis was reported as an effect of odontoblast cytodifferentiation disruption in vivo.29 DLX3 also participates in the control of fenretinide-mediated apoptosis in tumor cell lines.40 It has been proven that DLX3 participates in the negative regulation mechanism of apoptosis in DFCs and also regulates cell vitality/proliferation of DFCs (authors unpublished data). A reorganization of actin filaments in DFCs after DLX3 overexpression and the up-regulation of genes important for actin filament morphology regulation, such as MTSS1,41 proposes that DLX3 influences the cell shape of DFCs. Studies in MSCs suggest that stem cell commitment during differentiation is possibly regulated by cell shape and the alteration of their cytoskeletal components.42,43 Different studies have shown that BMP-2 induces the osteogenic differentiation of DFCs.20,44 DLX3 is induced by BMP-2 in diverse tissues such as osteoblasts.24,45 In hair follicles it was reported that colocalization of phospho-SMAD1/5/8 and DLX3 is consistent with a regulatory role of BMP signaling of DLX3 during hair morphogenesis.45 Recently, it was found that BMP-2 induces the expression of DLX3 during osteogenic differentiation of DFCs, while DLX3 induces also BMP-2 (authors unpublished data). Moreover, 50% of regulated genes are contrarily regulated in DFCs after BMP-2 supplementation and DLX3 overexpression.20 The authors hypothesized, therefore, that the TF DLX3 and BMP-2 (BMP signaling) control each other in DFCs via a feedback control. However, more
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Osteogenic differentiation OCN Runx2 Glucocorticoid receptor IL3R CyclinA2

Neutral differentiation

neurog1

Btbd6a Bmi-1 Gli3 BMP-7 Hox

ZBTB16

c-myc

Proapoptotic

Development

Fig 3 Proposed interaction partners of ZBTB16 in developmental and differentiation processes.

sophisticated studies are needed to clarify the detailed mechanisms and functions of DLX3-regulated pathways during the differentiation of DFCs.

aDDiTionaL TFs For oSTeogeniC DiFFerenTiaTion

Because typical osteogenic TFs such as RUNX2 or OSTERIX were not highly up-regulated during DFC differentiation,17 the TFs ZBTB16, NR4A3, TP53, and SP1 became new candidates for the evaluation of the process of osteogenic differentiation in dental stem cells.20,21 The following section summarizes cellular functions and current knowledge of these TFs for their putative roles in the molecular process of osteogenic differentiation.

ZBTB16
Zinc finger and BTB domaincontaining protein 16 (ZBTB16) consists of 673 amino acids (74 kDa), and its expression is induced by the glucocorticoid receptor. The DBD consists of nine Krppel-like H2-C2 zinc finger motifs.46 ZBTB16 was first discovered for its role in promyelocytic leukemia and named PLZF (promyelocytic leukemia zinc finger). As the result of a chromosomal reciprocal translocation (t(11;17) (q23;q21)) ZBTB16 is fused with the retinoic acid receptor-alpha gene, resulting in the inhibition of hematopoietic cell differentiation.47,48 Additional work with 310
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hematopoietic cells revealed that ZBTB16 can induce cell-cycle arrests in 32Dcl3 cells because of inhibition of the interleukin 3 receptor.49 Furthermore, ZBTB16 interacts with the vitamin D3 receptor,50 inhibits the cyclinA2 promoter,51 can induce apoptosis,52 and binds transcriptional repressors that inhibit transcription.53 ZBTB16 can also function as a transcriptional repressor, which can promote proliferation and inhibit differentiation or inhibit proliferation and promote differentiation, depending on the repressed target gene. The antiproliferative effect might be the result of a negative regulation of ZBTB16 on c-myc.54 In neural cells, ZBTB16 inhibits neurog-1 and therefore neural differentiation. Expression of Btbd6a inhibits ZBTB16 and induces neural differentiation.55 The participation of ZBTB16 in skeletal development has also been investigated. ZBTB16/ mice show impaired limb development, which might be because of an effect of ZBTB16 on BMP-7.49 In humans, ZBTB16 has also been shown to contribute to skeletal development. A loss of both ZBTB16 alleles causes severe skeletal abnormalities.56 Furthermore, ZBTB16 is involved in a hyperostosis disease. Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common disorder in Asian populations.57 Primary ligament cells isolated from OPLL patients showed increased ZBTB16 expression compared to control cells after induction of osteogenic differentiation.58 In the same work it was also shown that the up-regulation of ALP, Col1A1, and Runx2 after osteogenic differentiation is suppressed if ZBTB16 expression is inhibited by small interfering (siRNA). The ZBTB16 overexpressioninduced up-regulation of Runx2, col1A1, and OCN can also be inhibited by Runx2 siRNA treatment. Overexpression of Runx2 leads to up-regulation of col1A1 and OCN but not ZBTB16, which indicates that ZBTB16 might be an upstream regulator of Runx2 (Fig 3). The fact that BMP-2 induces the expression of ALP, Runx2, col1A1, and OCN but not ZBTB16 indicates that there might be an alternative signaling network for osteogenic differentiation.58 This is also supported by work with DFCs. It was shown that the up-regulation of osteogenic marker genes was more pronounced after BMP-2induced osteogenic differentiation, whereas Alizarin staining revealed stronger mineralization after dexamethasone-induced differentiation.20 Another finding that supports this hypothesis is that the BMP2 inhibitor noggin does not impair dexamethasoneinduced osteogenic differentiation.20 Furthermore, after transient overexpression of ZBTB16 in DFCs, the osteogenic marker genes OPN and OCN were up-regulated and ALP activity increased (authors unpublished data). All these data suggest an important function of ZBTB16 in dental stem cells.

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nr4a3
Nuclear receptor subfamily 4, group A, member 3 (NR4A3), a 626amino acid (68-kDa) protein also known as neuron-derived orphan receptor 1, belongs to the steroid-thyroid hormone-retinoid receptor superfamily/nuclear receptor family of intracellular TFs. Like all nuclear receptors, NR4A3 possesses an N-terminal ligandindependent AF-1 transactivation domain, a two zinc fingerscontaining DBD, and a C-terminal ligandbinding domain containing a ligand-dependent AF-2 transactivation domain.59,60 The DBD target site is the NGFI-B-responsive element (NBRE: AAAGGTCA motif).61 NR4A3 can be induced by a variety of stimuli, including growth factors, inflammatory stimuli, cytokines, peptide hormones, and cellular stress,62 and is involved in cell proliferation, differentiation, inflammation, metabolism, and apoptosis.59,63 To date, there is no evidence that NR4A3 plays a role in osteogenic differentiation. However, some observations of the authors working group have awakened interest in this nuclear receptor. NR4A3 is up-regulated in DFCs after osteogenic differentiation.64 Furthermore, NR4A3 is up-regulated after overexpression of the TFs TP53 and ZBTB16 in DFCs and down-regulated after TP53 and SP1 silencing in DFCs and SHED.65 Although there are no functional data for NR4A3 during osteogenic differentiation, the authors results support a putative role of NR4A3probably in combination with other TFs such as TP53in the process of differentiation.

Recently, first hints have been found that link TP53 to the osteogenic differentiation of DFCs.21,65 TP53 was not only slightly up-regulated after osteogenic differentiation; its DBD also is known to recognize the promoter of 35% of all regulated genes during osteogenic differentiation.21 Additionally, osteogenic marker genes are up-regulated after a transient overexpression of TP53 in DFCs and SHED.65 Contradictory results from investigations of the effects of TP53 on the osteogenic differentiation of BMMSCs76,77 might be a result of the differences of dental stem cells because of their ectomesenchymal origin. The osteogenesisinhibiting properties of TP53 that were found in the aforementioned publications were exerted by a downregulation of Runx2, a factor whose up-regulation might be not important for the dexamethasone-induced differentiation of dental stem cells.17,20 Furthermore, TP53 was shown to have a positive influence on the osteogenic differentiation of muscle-committed MSCs.78,79 Additionally, it was shown for some stem cells that TP53 inhibits self-renewal,8082 and the generation of induced pluripotent stem cells is facilitated in the absence of TP53,83 which also supports the idea of a role of TP53 in differentiation.

Sp1
The TF specificity protein 1 (SP1) belongs to the specificity protein/Krppel-like factor family of TFs and contains three C2H2-type zinc fingers. SP1 consists of 785 amino acids (81 kDa) and binds GC-rich regions of its target DNA (consensus sequence 5-(G/T) GGGCGG(G/A)(G/A)(C/T)-3). The genes regulated by SP1 are involved in many biologic processes, including cell growth, apoptosis, differentiation, and immune responses.84,85 SP1 undergoes pronounced posttranslational modifications such as phosphorylation, sumoylation, proteolytic cleavage, glycosylation, and acetylation. Glycosylation and phosphorylation are important for the nuclear location of SP1. This state is promoted by insulin, whereas cytoplasmic location of SP1 is promoted by glucagon.86 SP1 regulates Col11A2 in human Saos-2 osteoblastic cells.87 Furthermore, it was shown that SP1 is important for the recruitment of SP7 (osterix) to the OCN promoter in rats.88 Zhang et al showed that SP1 is involved in Runx2 promoter activity in the mouse osteoblastic cell line MC3T3-E1.89 Felthaus et al were able to show that overexpression of SP1 leads to an up-regulation of col1A1 in SHED.65 No other osteogenic marker genes were regulated and the ALP activity did not significantly change after SP1 overexpression. This is in accordance with the fact that genes that are regulated during osteogenic differentiation and have promoter binding sites for SP1 were down-regulated.21 After siRNA inhibition of SP1, however, OPN and Runx2
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Tp53
TP53 consists of 393 amino acids (43.7 kDa). The DBD of TP53 contains a central zinc ion and several arginine residues. TP53 is well known for the effects it exerts on the regulation of cell cycle progress and apoptosis. TP53 is a highly unstable protein that is rapidly degraded in cells. As a result of cellular stress, such as DNA damage or oxidative stress, TP53 is posttranslationally stabilized, accumulates in the cell, and undergoes a conformational change as a result of N-terminal phosphorylation.66,67 The activated TP53 induces the expression of p21, which causes a cell cycle arrest. If the cell fails to repair the damaged DNA, TP53 accumulates further and induces the expression of genes of the Bcl2 family, which eventually leads to apoptosis.6870 It therefore functions as a tumor suppressor. The TP53 gene is mutated in 50% of all human cancers.71 However, recent research has focused on the role TP53 might play in differentiation and stem cell maintenance. As a reaction to DNA damage in embryonic stem cells, TP53 rather induces differentiation by inhibiting nanog than cell cycle arrest. Furthermore, TP53 prevents the dedifferentiation of progenitor cells67,72 and the generation of induced pluripotent stem cells.7375

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were down-regulated in DFCs and SHED. The authors suggest that SP1 in dental cells has a modulatory effect on the process of osteogenic differentiation and that SP1 is also involved in the regulation of cell proliferation.

ouTLook
Recently, gene expression profiles were recorded from the dental stem cells SHED, DFCs, and dNCPCs, as well as the MG63 osteosarcoma cell line as a nondental control after osteogenic differentiation (Gtz et al, unpublished data). Additionally, Felthaus et al have already investigated gene expression profiles of DFCs after transient overexpression of DLX3, ZBTB16, TP53, or SP165 (Felthaus et al, unpublished data). These microarray data support the hypothesis that dental stem cells share some special characteristics during osteogenic differentiation and that the TFs DLX3, ZBTB16, and TP53 play important roles in cell differentiation. Here, several genes are generally regulated and have turned out to be interesting candidates for the evaluation of the mechanisms of osteogenic differentiation in dental stem cells, especially in DFCs. For example, stanniocalcin 1 (STC1), which is involved in cellular calcium ion homeostasis and associated with osteogenic differentiation,90 is up-regulated in DFCs after overexpression of DLX3, ZBTB16, and TP53 but not after overexpression of SP1. Furthermore, STC1 is up-regulated after osteogenic differentiation in SHED, DFCs, and dNC-PCs but down-regulated in MG63. This indicates that STC1 might be important for osteogenic differentiation in dental stem cells and that such TFs as DLX3, ZBTB16, and TP53 play important roles in this possible mechanism. The expression profiles of many other genes confirm the finding that SHED, DFCs, and dNC-PCs have common characteristics that they do not share with MG63 cells and that overexpression of DLX3, ZBTB16, and TP53 have common characteristics that they do not share with the transcriptome after SP1 overexpression65 (Felthaus et al, unpublisheddata).AmongtheseareBMP-2,angiopoietinlike 4 (ANGPTL4), a gene involved in cell differentiation, and growth differentiation factor 15 (GDF15), a gene involved in the transforming growth factor-beta signaling pathway. For single genes, the molecular function in the process of osteogenic differentiation is under investigation, but (for example) GDF15 and ANGPTL4 are interesting new targets for further studies about molecular processes in dental stem cells. Some concerns need to be addressed when working with TFs for tissue engineering applications. For example, a stable transfection of TFs using a virus is accompanied with the risk of severe changes in the 312
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cell genome. This can result in the loss of their multipotency or the development of tumorigenic characteristics. On the other hand, a transient transfection, as well as the direct application of TFs in the form of proteins, might not be sufficient to differentiate the cells in the favored direction. Whereas this problem can be solved in cell culture by repeated application of expression vectors or proteins, the need for a scaffold and the resulting three-dimensional growth might limit this treatment for tissue engineering purposes.

reFerenCeS
1. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs those from other sources: Their biology and role in regenerative medicine. J Dent Res 2009;88:792806. 2. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 2000;97:1362513630. 3. Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA 2003;100:58075812. 4. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364:149155. 5. Morsczeck C, Gotz W, Schierholz J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol 2005;24:155165. 6. Sonoyama W, Liu Y, Yamaza T, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: A pilot study. J Endod 2008;34:166171. 7. Degistirici O, Jaquiery C, Schnebeck B, et al. Defining properties of neural crest-derived progenitor cells from the apex of human developing tooth. Tissue Eng Part A 2008;14: 317330. 8. Karaz E, Demircan PC, Sag lam O, Aksoy A, Kaymaz F, Duruksu G. Human dental pulp stem cells demonstrate better neural and epithelial stem cell properties than bone marrowderived mesenchymal stem cells. Histochem Cell Biol 2011;136:455473. 9. Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M, Magloire H. Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif Tissue Int 2000;66:129138. 10. Sakai VT, Zhang Z, Dong Z, et al. SHED differentiate into functional odontoblasts and endothelium. J Dent Res 2010; 89:791796. 11. DSouza RN, Cavender A, Sunavala G, et al. Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo. J Bone Miner Res 1997;12:20402049. 12. Grummt I. Regulation of mammalian ribosomal gene transcription by RNA polymerase I. Prog Nucleic Acid Res Mol Biol 1999;62:109154. 13. Willis IM. RNA polymerase III. Genes, factors and transcriptional specificity. Eur J Biochem 1993;212:111. 14. Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J 2004;23:40514060. 15. Brivanlou AH, Darnell JE Jr. Signal transduction and the control of gene expression. Science 2002;295:813818.

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