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Matrix and TGF-[Beta]-Related Gene Expression During Human Dental Pulp Stem Cell (DPSC) Mineralization

July 3, 2007

By Liu, Jun Jin, Taocong; Chang, Syweren; Ritchie, Helena H; Et al

Abstract We have recently reported the induction of dental pulp stem cells (DPSCs) into dentin-secreting odontoblast-like cells after stimulation by isolated dentin matrix components, thus mimicking the nature of tissue regeneration seen after tooth disease and injury. After confluency, the cells were further cultured for 21 d in the 10% fetal bovine serum (FBS) Dulbecco’s modified Eagle’s medium (DMEM) (control), and in this medium, with the addition of dentin extract (DE) and the mineralization supplement (MS) of ascorbic acid and beta-glycerophosphate (treatment). To identify genes associated with this process, specimens were analyzed with a HG-U133A human gene chip and Arrayassist software. A total of 425 genes, among them 21 matrix and eight TGF-beta-related genes, were either up- or downregulated in the experimental group in which the cells showed odontoblast-like differentiation and mineralization. Expression of selected genes was further confirmed by real-time polymerase chain reaction (PCR) analysis. Of the extracellular matrix (ECM)-related genes, two types of collagen genes were upregulated and seven others downregulated. Other ECM-related genes, for example fibulin-1, tenascin C, and particularly thrombospondin 1, were upregulated, and fibulin-2 was downregulated. Most noticeably, the matrix metalloproteinase 1 was induced by the treatment. In the TGF-beta superfamily, upregulation of the type II receptor, endoglin, and growth/differentiation factor 5 was coordinated with the downregulation of activin A, TGF-beta2, and TGF- beta1 itself. This study identifies the matrix and TGF-beta-related gene profiles during the DPSC cell mineralization in which several genes are reported for the first time to be associated with this process, thus greatly expanding our molecular knowledge of the induced disease repair process. Keyword Extracellular matrix * Transforming growth factor * Microarray * Stem cell * Mineralization

Introduction

Recently, a distinct population of human postnatal dental pulp stem cells (DPSCs) have been described (Gronthos et al. 2002; Shi et al. 2005) and similar cells have been isolated from shed deciduous teeth (Miura et al. 2003). These postnatal stem/progenitor cells have offered us promising possibilities either for the regeneration from resident populations in the pulp or for the engineering tissue constructs.

The key to exploitation of these DPSCs will be understanding the signaling events involved in their differentiation to odontoblasts for dentinal matrix protein secretion and the molecular processes orchestrating these cellular events. Recently, we have described the in vitro induction of these DPSCs into odontoblast-like cells after treatment with an EDTA-soluble dentin extract (DE) and a mineralization supplement (MS). Within this previous study, we detected a significant upregulation of the gene expression of sialophosphoprotein (Dspp), an important phenotypic marker of odontoblasts, under circumstances of DE+MS treatment (Liu et al. 2005). Such preparations of dentin matrix components include a cocktail of growth factors and bio-active molecules sequestered in the dentin, and their action on the DPSC cells was used to mimic the molecular events in dental tissue regeneration after injury. Odontoblast-like cell differentiation from postnatal stem cells resident in the pulp (DPSCs) is signaled after release of growth factors and bio-active molecules from the dentin matrix after injury (Smith and Lesot 2001). The availability of this experimental system of DPSCs and dentin matrix components provides a model in which the changes in gene expression of DPSCs before and after differentiation may be probed and compared. Such studies will allow a better understanding both of the nature of the phenotypic changes in the DPSCs as they differentiate into odontoblasts and the manner in which their functionality changes. Characterization of these features of the DPSCs is fundamental to harnessing their potential for use in tissue regeneration and engineering applications.

Despite numerous reports on the composition of dentin extracellular matrix (ECM) including collagenous (i.e., collagen types I, III, IV, and V) and noncollagenous ECM components (i.e., dentin phosphoprotein, dentin sialoprotein, decorin, and biglycan) and the putative roles of some of the components, no specific molecules appear to be unique to this tissue. Instead, there appears to be a profile of molecules that characterizes this tissue and contributes to its unique morphological structure and function. The aim of the present study was to investigate how gene expression changes as DPSCs differentiated into odontoblasts and mineralize under the influence of a preparation of dentin ECM components mimicking the events of dentin regeneration after tissue injury, with particular focus on ECM and TGF-p-related gene patterns.

Materials and Methods

Cell culture and treatment with dentin extracts. A soluble extract of porcine immature root dentin was prepared by incubation in 10% EDTA, pH 7.2 containing protease inhibitors at 4[degrees]C for 10 d followed by dialysis against water and lyophilization. The DPSC cells used in this study were kindly donated by Dr. S. Shi (NIH). DPSC cells at passage 8 were subcultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 units/ ml of penicillin and 100 [mu]g/ml of streptomycin, at a seeding density of 0.8 x 10^sup 6^, in 60-mm culture dishes. The cultures were maintained at 37[degrees] C in a humidified atmosphere containing 5% CO2 and 95% air. After growth for 2 d till confluency, the cells were further cultured for 21 d in the presence of the EDTA- soluble dentin extract (DE, 10 [mu]g/ml), with the addition of the mineralization promoting supplement (MS) of ascorbic acid (AA, 50 [mu]g/nll) and beta-glycerophosphate (beta-GP, IO mM). The medium and the supplements were changed every other day. After 21 d, the cells were harvested and used for the following exerimente.

RNA isolation and reverse transcription. Total cellular RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The RNA was treated with the RNase-free DNase Set (Qiagen) during isolation. Two raicrograms of total RNA were used for cDNA synthesis with the TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA) in a 100-[mu] reaction volume.

Gene microarray analysis. A human gene chip HGU133A (Affymetrix, Santa Clara, CA), containing approximately 14,500 well- characterized human genes, was used. Ten micrograms of total RNA (OD 260/280>/=1.80) was quantitatively amplified and biotin-labeled according to GeneChip Expression Analysis Technical Manual. Briefly, RNA was converted to double-stranded complementary DNA (cDNA) using Superscript II RT (Invitrogen, Carlsbad, CA) with a T7-(dT)24 primer (Affymetrix). Then, the cDNA was used in an in vitro transcription reaction in the presence of biotin-modified ribonucleotides (Enzo, Farmingdale, NY) to produce large amounts of singlestranded RNA. The biotin-labeled RNA was fragmented and 10 [mu]g of them hybridized to the gene chip HG-U133A at 45[degrees]C for 16 h. Labeled bacterial RNAs of known concentration were spiked in hybridization to generate an internal standard and to allow normalization between chips. Chips were washed, stained with streptavidin R-phycoerythrin (Molecular Probes, Eugene, OR). After scanning the chips, the data were analyzed by using Affymetrix GeneChip-related software including Microarray Suite, Data Mining Tool, and Arrayassist. The expression change of at least twofold with a p value smaller than 0.05 was set as the “cutoff value for the transcripts being differentially expressed in the treatment and control group, and used in determination of significant differences for further studies. Duplicate chips were used in this microarray study.

Real-time polymerase chain reaction (PCR) quantitation. Quantitative real-time PCR analysis was carried out using an ABI 7700 sequence detector (Applied Biosystems). The assay-on-demand human gene products, which contain two unlabeled PCR primers and a FAM(TM) dye-labeled TaqMan(R) minor groove binder (MGB) probe, were used for the quantitative detection of the following gene markers. COL4A1 (Hs00266237_ml), COL13A1 (Hs00193225_ ml), FBLNl (Hs00242546_ml), FBLN2 (Hs00157482_ ml), TNC (Hs00233648_ml), MMPl (Hs00233958_ml), TFPI2 (Hs00197918_ml), GDF5 (Hs00167060_ml), INHBA (HsOOl 70103_ml), TGFBR2 (Hs00559661_ ml), THBSl (Hs00170236_ml) (Table 1). Real-time PCR was performed using the TaqMan Universal PCR Master Mix Kit (Applied Biosystems). Briefly, a 30-[mu]l PCR reaction was prepared with 1-[mu]l cDNA (RT product) and 1.5-[mu]l mixture of specific MGB probe and primers as mentioned above. The thermal conditions were: 50[degrees] C 2 min, 95[degrees] C 10 min followed by 45 cycles of 95[degrees] C 15 s and 60[degrees] C 1 min. The target gene expression was normalized to the housekeeping gene beta-actin. Relative gene expression values were calculated using a standard curve method.

Morphological observation. Phase-contrast microscopy was used to observe cell growth, formation of cell multilayers and cell nodules.

Results

Induction of DPSC cell differentiation and mineralization by amino acid (AA), beta-glycerophosphate (beta-GP), and dentin extracts. Twenty-one days after cell confluency, obvious cell nodule formation and mineral deposition within and around the cell nodules were observed by phase contrast microscopy in the MS+DE treatment group. Cells were arranged in parallel, aligned perpendicular to the cell nodule with elongated cell process. Cells in control cultures hi the absence of MS+DE were still fibroblastic in morphology, showed no ordered arrangement nor mineral deposition at the same experimental tune period (Fig. I). Mineralization in the experimental group was further confirmed by von Kossa staining after continuous culture for 28 d {Liu et al. 2005). No mineralization occurred in control cultures in the absence of MS (+-DE). Differentially expressed matrix and TGF-j3-related genes during DPSC cell mineralization analyzed by microarmy study. Human gene chips HG- U133A, containing approximately 14,500 well-characterized human genes, were used for the initial fishing and detection of the differentially expressed matrix and TGFbeta-related genes during DPSC cell mineralization after 21 d. The microarray data were analyzed using Affymetrix GeneChip-related software including Microarray Suite, Data Mining Tool, and Arrayassist. With Arrayassist, differential expression of at least twofold with a p value smaller than 0.05 was used to determine the significant differences for further studies. Among the 425 differentially expressed genes regulated in the MS+DE treatment, 21 matrix and eight TGFbeta-related genes were either down or upregulated as the mineralization occurred. Of the extracellular-matrix-related genes, two types of collagen genes were upregulated and seven others downregulated. Other ECM-related genes, for example ftbulin-1, matrix GIa protein (MGP), tenascin C, and particularly thrombospondin 1 were upregulated, and fibulin-2, biglycan, and tetranectin were downregulated. Most noticeably, matrix metalloproteinase 1 was induced by the treatment. MMP-I inhibitors, such as TIMP-3 and TFPI2, were also upregulated in this process. Among the TGF-beta superfamily members, upregulation of the type II receptor, type III receptor-endoglin, and growth/differentiation factor 5 were coordinated with the downregulation of activin A, TGF- beta2, and TGF-beta1 itself (Fig. 2, Table 2).

Real-time PCR quantitaiion. To verify the results obtained from the microarray analysis, quantitative realtime PCR analysis was carried out for 11 selected transcripts. The target gene expression was normalized to the housekeeping gene beta-actin. Relative gene expression values were calculated using a standard curve method (Fig. 3). The differential fold changes analyzed by both microarray and quantitative real-time PCR were consistent with each other (Tables 3 and 4). Noticeably for MMPl expression, the microarray analysis showed the induction of MMPl during mineralization, which is in accord with the >2000 -fold upregulation detected by the real- time PCR quanti talion.

Discussion

In this study, we have used an EDTA-soluble matrix extract of dentin, which includes a cocktail of growth factors and bio-active molecules sequestered in the dentin, to treat the DPSC cells to mimic the molecular events in dental tissue regeneration after injury. Odontoblast-like cell differentiation from postnatal stem cells resident in the pulp (DPSCs) is signaled after release of growth factors and bio-active molecules from the dentin matrix (Smith and Lesot 2001). Odontoblast-like cell differentiation and subsequent mineralization were induced by treating the cells with DE+MS for 21 d; obvious cell nodule formation and mineral deposition within and around the cell nodules were observed after this treatment. Cells were arranged in parallel, aligned perpendicular to the cell nodule with elongated cell processes characteristic of odontoblasts. Cells in control cultures were still fibroblastic in morphology, showing no ordered arrangement or mineral deposition at the same experimental time period indicating a direct effect of the DE+MS treatment on odontoblast differentiation. Odontoblast differentiation and dentin mineralization during either primary or reparative dentin formation involves not only the participation of various molecules including growth factors, receptors, and extracellular matrix components for cell signaling, but also complex regulatory mechanisms for these signaling events. Defining the roles of molecules that orchestrate dentin matrix mineralization and their regulatory mechanisms are critical steps in better understanding the molecular events underlying this process and the clinical exploitation of corresponding dental pulp progenitor cells in regenerative therapies. In the present study, we have been able to identify 21 matrix and eight TGF-beta-related genes that were differentially expressed during the cell mineralization.

Among the collagenous components, two collagen genes were upregulated and seven others downregulated. The results of our study, for the first time, detected and then confirmed by real-time PCR the expression of type XIII collagen in human denial pulp cells and its upregulation during DPSC cell differentiation and mineralization. Type XIII collagen has been reported to be closely involved in cell adhesion, cell adhesion-dependent functions, and multiple cell-matrix interactions (Tu et al. 2002; Vaisanen et al. 2004). Recently, the type XIII collagen has been found to be strongly associated with the increased bone formation in a transgenic mice study. (Ylonen et al. 2005) Thus, in our study, the upregulation of type XIII collagen may also be related with the mineralized nodule formation after the cell differentiation. The expression of type X collagen, which had been previously observed to be strongly distributed in the developing human enamel organ and ameloblasts (Felszeghy et al. 2000), has also been detected for the first time in DPSCs and was significantly upregulated after the differentiation of these cells. In accord with the loss of the basement membrane after odontoblast differentiation during physiological tooth development, one of the major components of the basement membrane, type IV collagen, was significantly downregulated with DPSC differentiation after DE+MS treatment.

Among the noncollagenous ECM-related genes, tenascin C and particularly thrombospondin 1, which is highly expressed in odontoblasts (Ueno et al. 1998), were significantly upregulated during differentiation and mineralization in the DPSCs. Both FGF-4 and TGF-beta, which would be present hi our dentin matrix extract, have been previously shown to stimulate tenascin C expression in El2 mouse dental mesenchyme (Sahlberg et al. 2001). A high level of THBSl mRNA expression has only been detected in odontoblasts and not in the dental pulp or gingival tissue (Ueno et al. 1998). The role of THBSl in binding and activating TGF-beta is well established (Lawler 2000; Daniel et al. 2004) and its upregulation in odontoblasts correlates with the expression of TGF-beta isoforms in these cells (Sloan et al. 2000). It is not surprising to see that the small leucinerich proteoglycan (SLRP) member, biglycan (BGN), was inhibited after differentiation of DPSCs into odontoblasts, as BGN has been suggested to have an adverse effect on the dentin mineralization process (Sreenath et al. 2003; Fanchon et al. 2004). The upregulation of MGP during DPSC cell mineralization may be related to its role in the prevention of hypermineralization (Hashimoto et al. 2001; Murshed et al. 2004). Interestingly, the stimulation of fibuluvl and inhibition of fibulin-2 in our experiment may be associated with multiprotein complex formation with die cell adhesion molecule fibronectin. Thrombospondin 1 may also participate in multiprotein complex formation.

Recently, matrix metalloproteinases (MMPs) have been suggested to play a central role in the remodeling of toothspecific matrix and to maintain a favorable local microenvironment for the survival of odontoblasts (Randall and Hall 2002; Palosaari et al. 2003; Ogbureke and Fisher 2004; Fanchon et al. 2004; Bourd-Boittin et al. 2005). The activity of pro and active forms of MMPs can be regulated by binding to different tissue inhibitor of metalloproteinases (TIMPs). Tissue factor pathway inhibitor 2 (TFP12), a serine proteinase inhibitor, has also been shown to inhibit the activity of MMP-I, – 2, -9, and MMP-13(Baker et al. 2002). Notably in our study, MMP-I was induced by the treatment, and real-time PCR confirmed a >200 times upregulation of this molecule. MMP-I inhibitors, such as TIMP- 3 and TFPI2, were also up regulated in this process. MMP-I, TIMP-I, – 2 and -3 have been detected both in odontoblasts and pulp tissue (Sulkala et al. 2002; Palosaari et al. 2003). In addition to the inhibitory effects on MMP-I, TIMP-3 may also show intrinsic functional roles in the differentiation of odontoblasts as suggested from tooth development studies (Yoshiba et al. 2003). MMPl expression has been shown to be upregulated by cytokines (Tamura et al. 1996; Lin et al. 2001; Shin et al. 2002) and another MMP, MMP- 9, was significantly upregulated by TGF-beta-1 {Palosaari et al. 2003). TFPI2 has been reported to inhibit ProMMP-1 activation and MMP-1 activities (Rao et al. 1999; Baker et al. 2002). Thus, in the present study, the induction of MMP-I together with the balanced upregulation of its inhibitor TIMP-3 and TFPI2, may play critical roles in remodeling the extracellular matrix to create a favorable microenvironment for DPSC cell differentiation and mineralization to occur.

Among the TGF-beta-related transcripts, we have detected the upregulation of the type II receptor, type HI receptorendoglin and growth/differentiation factor 5, and the corresponding downregulation of activin A, TGF-beta2, and TGF-beta1 itself (Fig. 2, Table 2). TGF-beta signal transduction usually first involves the binding of TGF-beta to its Type II receptor (TGF-BR2), then the activation of the Type I receptor (TGF-BRO, or combined with the participation of the accessory or Type III receptor (endoglin). TGF- BRl activation results in the phosphorylation of intracellular messengers such as Smad proteins for signal transduction (He et al. 2001). GDF5, which has close structural relationship with bone morphogenetic proteins (BMPs), has been reported to be expressed in the odontoblast layer in bovine incisor tooth germs (Morotome et al. 1998) and induced the arrest of cell growth in the GI phase (Nakahara et al. 2003), which may be reflected in the Growth Arrest Specific 1 (GASl) upregulation in our DPSC treatment group. In addition, the FK506 binding protein IA (FKBPlA), another TGF-beta member, interacts with and regulates the TGF-BRl. This is the first report of this molecule in dental pulp cells and its upregulation during odontoblast differentiation. Activin A, another TGF-beta family member, was shown to inhibit the early differentiation of the fetal rat calvarial cells (Ikenoue et al. 1999). The negative feedback regulation mechanism may be attributed to the downregulation of TGF-beta2 and TGF-beta1. Clearly, a complex series of interactions involving TGF-beta family members are associated with the DPSC cell mineralization process. In summary, we have identified a number of differentially expressed genes after the differentiation of DPSCs into odontoblast-like cells and becoming mineralized. This delicately controlled pattern of changes in gene expression involves ECM components, MMPs and their inhibitors, which are closely related with matrix remodeling, and TGFa signaling complexes. Identification of these molecules will now allow functional approaches to studies on their roles during odontoblast development and matrix secretion and mineralization.

Acknowledgment This study was supported by NIH grants DE 12899 and DE 11442. We thank Dr. Jacques E. Nor for his helpful discussion and critical review of this manuscript.

Received: 29 March 2007 / Accepted: 2 April 2007 / Published online: 22 May 2007 / Editor: J. Denry Sato

(c) The Society for In Vitro Biology 2007

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J. Liu * T. Jin * S. Chang * H. H. Ritchie * B. H. Clarkson (*)

Department of Cariology, Restorative Sciences and Endodontics,

School of Dentistry, University of Michigan,

1011 North University,

Ann Arbor, MI 48109-1078, USA

e-mail: bricla@umich.edu

A. J. Smith

Oral Biology, School of Dentistry, University of Birmingham,

St. Chad’s Queensway,

Birmingham B4 6NN, UK

Copyright Society for In Vitro Biology Mar/Apr 2007

(c) 2007 In Vitro Cellular & Developmental Biology; Animal. Provided by ProQuest Information and Learning. All rights Reserved.




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