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J Dent Res 83(6): 496-499, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Dentonin, a Fragment of MEPE, Enhanced Dental Pulp Stem Cell Proliferation

H. Liu1,2, W. Li1, C. Gao1, Y. Kumagai3, R.W. Blacher3, and P.K. DenBesten1,*

1 Box 0640, University of California, San Francisco, CA 94143-0640, USA;
2 Peking University School of Stomatology, Beijing, China; and
3 Acologix, Emeryville, CA, USA;

* corresponding author, pkdb{at}itsa.ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix extracellular phosphoglycoprotein (MEPE) is a SIBLING protein, found in bone and dental tissues. The purpose of this study was to determine whether a 23-amino-acid peptide derived from MEPE (Dentonin or AC-100) could stimulate dental pulp stem cell (DPSC) proliferation and/or differentiation. DPSCs were isolated from erupted human molars, and the mitogenic potential of Dentonin in DPSCs was measured by BrdU immunoassay and cell-cycle gene SuperArray. Differentiation of DPSCs with Dentonin was characterized by Western blot and by osteogenesis gene SuperArray. Dentonin enhanced DPSC proliferation by down-regulating P16, accompanied by up-regulation of ubiquitin protein ligase E3A and human ubiquitin-related protein SUMO-1. Enhanced cell proliferation required intact RGD and SGDG motifs in the peptide. This study shows that Dentonin can promote DPSC proliferation, with a potential role in pulp repair. Further studies are required to determine the usefulness of this material in vivo.

KEY WORDS: MEPE • dental pulp stem cells • proliferation • Dentonin • AC-100


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dentin mineralization is believed to be directed in part by non-collagenous proteins (Butler and Ritchie, 1995). Matrix extracellular phosphoglycoprotein (MEPE) is a newly identified, non-collagenous, protein that has been found in human bone and dental tissues (Rowe et al., 2000; Argiro et al., 2001; MacDougall et al., 2002). MEPE is a related member of the bone/dentin SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family, which includes dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP1), osteopontin (OPN), and bone sialoprotein (BSP) (Fisher and Fedarko, 2003). The genes coding for SIBLINGs are clustered in a defined region in chromosome 4q (4q21.1 for MEPE), the critical loci for dentinogenesis imperfecta types II and III, and dentin dysplasia type II (MacDougall et al., 2002). MEPE is expressed in bone by fully differentiated osteoblasts, and is associated with an inhibition of bone formation and mineralization (Gowen et al., 2003; Quarles, 2003).

The search for the bioactive fragment of MEPE resulted in the synthesis of Dentonin (AC-100, Acologix, Emeryville, CA, USA), a 23-amino-acid residue that corresponds to region 242–264 of human MEPE. Dentonin peptide (TDLQERGDNDISPFSGDGQPFKD) is derived from the conserved sequences in the center of MEPE, and contains the RGD integrin-binding motif, the SGDG glycosaminoglycan-binding motif, and was shown to promote bone formation and osteoblast proliferation (Hayashibara et al., 2004). Since dentin and bone have many similarities in matrix components, it is possible that MEPE and Dentonin also function in dentin formation.

During pulp injury, a subgroup of precursor cells in the pulp may differentiate into odontoblasts to form reparative dentin. Recently, Gronthos and colleagues (Gronthos et al., 2000, 2002) have identified a population of post-natal dental pulp stem cells (DPSCs) in human dental pulp, which may be one of the sources of these precursor cells. In vitro and in vivo studies have shown that DPSCs can undergo differentiation into odontoblast-like cells and regenerate a dentin-pulp-like complex when transplanted into nude mice (Gronthos et al., 2000, 2002; Batouli et al., 2003). In this study, we investigated the role of Dentonin in dental pulp stem cell (DPSC) proliferation and differentiation.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DPSC Isolation
DPSCs were isolated as described by Gronthos et al.(2000), following approval by the institutional review board. Cells were released from human pulp tissues by treatment with collagenase type I (3 mg/mL) and dispase (4 mg/mL), and were seeded into 10-cm plates (Costar, Cambridge, MA, USA) at low density (0.05 x 105/plate). Rapidly growing single colonies were isolated with the use of a cloning cylinder, and were expanded with alpha-modified Eagle’s Medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 20% fetal calf serum (Equitech-Bio Inc., Kerrville, TX, USA), 100 µM L-ascorbic acid 2-phosphate (WAKO, Tokyo, Japan), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Biofluids Inc., Rockville, MD, USA), and incubated at 37°C in 5% CO2.

Effect of Dentonin on Cell Proliferation
    Chemiluminescence Immunoassay
Cells were grown in clear black 96-well plates until they were 60% confluent. The cells were then starved in serum-free media for 24 hrs. Dentonin (TDLQERGDNDISPFSGDGQPFKD), RGD-altered peptide (TDLQEDRGNDISPFSGDGQPFKD), and SGDG-altered peptide (TDLQERGDNDISPFGDGSQPFKD) (Acologix Inc., Emeryville, CA, USA) were added in triplicate at 0, 0.05, 0.1, 0.25, 0.5, 0.75, and 1 µg/mL media, respectively. Cells were maintained in this medium for 24 hrs, and cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation with the use of an ELISA BrdU kit (Roche, Mannheim, Germany). Luminescence was detected on a SPECTRAmax Microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) at an emission of 425 nm. Statistical significance between groups was tested by ANOVA with Tukey’s post-test analysis.

    Cell-cycle Gene SuperArray
A GEArray Q series human cell-cycle gene array kit was obtained from SuperArray Inc. (Bethesda, MD, USA). This array included 96 genes involved in cell-cycle regulation. Included in this array are CDKs and CDK-modifying proteins, including cyclins, CDK inhibitors, and CDK kinases as well as genes in the SCF and APC ubiquitin-conjugation complexes. (See www.superarray.com for details.)

DPSCs were plated at a density of 1.5 x 104/mL on 10-cm dishes (Primaria, Falcon), were cultured until 60% confluence, and then serum-starved for 24 hrs. The cells were divided into two groups. Dentonin was added into one group at 1 µg Dentonin/mL medium, while the other group served as a control without Dentonin. After 48 hrs, total RNA was isolated with the use of a RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA), and 5 µg RNA was used as a template to generate Biotin-16-dUTP-labeled cDNA probes according to the manufacturer’s instructions. The cDNA probes were denatured and hybridized at 60°C with the SuperArray membrane, which was washed and exposed with the use of a chemiluminescent substrate.

To analyze the SuperArray membrane, we scanned the x-ray film and imported it into Adobe Photoshop as a TIFF file. The image file was inverted, and the spots were digitized with the use of ScanAlyze software (shareware, http://rana.lbl.gov/EisenSoftware.htm), and normalized by subtraction of the background as the average intensity value of 3 spots containing plasmid DNA (PUC18). The averages of 2 GAPDH or 4 cyclophilin A spots were used as positive controls and set as baseline values with which the signal intensity of other spots was compared. Using these normalized data, we compared the signal intensity from the membranes using the GEarray analyzer program (SuperArray Corp., http://www.superarray.com).

Effect of Dentonin on Cell Differentiation
    Western Blot
Cells were cultured in two groups, one with 1 µg/mL Dentonin and the control group without Dentonin. The cells were harvested in SDS lysis buffer at either 90% confluence, at complete confluence, or at the initiation of mineralization. The samples were sonicated, boiled for 1.5 min, and centrifuged at 10,000 rpm for 5 min. The protein in the supernatant was measured by a BioRad Coomassie Blue protein assay (Bio-Rad Laboratories, Hercules, CA, USA) and adjusted to the same concentration. Equal amounts of protein were loaded onto a 15% polyacrylamide gel and separated by electrophoresis. Western blots were done with anti-DSP (1:200 dilution of LF-151 (Gronthos et al., 2000), or anti-actin (Santa Cruz Biotechnology). Positive bands were detected by an ECL chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA).

    Osteogenesis Gene SuperArray
SuperArray membranes containing 96 osteogenesis-related cDNAs were purchased from SuperArray Inc. (Bethesda, MD, USA). Membranes were exposed to probes generated from mRNA of either control or Dentonin-treated DPSCs, according to the same protocol as described above for the cell-cycle SuperArray analysis.

    Mineral Induction and Quantitative Alizarin Red Staining
Cells were cultured in the presence of 50 µg/mL ascorbic acid, 10 mM ß-glycerophosphate, and 10 nM dexamethasone (Sigma) to induce mineral formation. Cells were grown either with 1 µg/mL Dentonin in the media, or without Dentonin. Nodule formation was observed after 3 wks of culture, and quantitative Alizarin Red staining was performed as described by Stanford et al.(1995). Briefly, the samples were fixed with 4% paraformaldehyde overnight, rinsed in PBS, and stained for 10 min with 40 mM Alizarin Red solution, pH 4.2, at room temperature. The cells were then rinsed 5 times with water, followed by a 15-minute wash with PBS to reduce non-specific staining. We quantitated the amount of Alizarin Red stain bound to the mineral in each dish by de-staining each sample in 10 mM sodium phosphate containing 10% cetylpyridinium chloride, pH 7.0, for 15 min at room temperature. The amount of Alizarin Red stain in the de-staining solution was determined by measurement of the absorbance of the solution at 562 nm, and comparison with standard solutions with known concentrations of Alizarin Red stain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DPSC differentiation into odontoblasts was characterized by increasing expression of DSP, as shown in Fig. 1Go. The Western blot results showed that DPSCs secreted low levels of DSP protein prior to confluence. As the cells reached confluence, the amount of DSP increased, and remained high as nodules began to form.



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  		Figure 1. Western blot of DSP separated by SDS PAGE. Equal amounts of protein from pre-confluent DPSCs (PC), confluent (C) DPSCs, and cells with mineralizing nodules (M) were loaded into each lane. There was no detectable effect of Dentonin on the relative level of DSP secreted at each stage of culture.

 
Dentonin was evaluated for mitogenic activity on DPSCs. Addition of Dentonin (0.05 to 1 µg/mL) resulted in a significantly increased rate of cell proliferation (p <= 0.05), with an optimal concentration between 0.5 µg/mL and 0.75 µg/mL of Dentonin (Fig. 2AGo). Alteration of the SGDG motif resulted in significantly reduced mitogenic activity (p <= 0.05) relative to the intact Dentonin peptide, at 0.5 and 0.75 µg/mL. Alteration of the RGD motif resulted in greater variability of response relating to increased concentrations of Dentonin and DPSC proliferation (Fig. 2BGo).



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  		Figure 2. The effect of Dentonin on DPSC proliferation shows: (A) the rate of cell proliferation in Dentonin-treated cells increased relative to the control group (p <= 0.05); and (B) DPSC proliferation was most enhanced with Dentonin peptide, and was reduced when either the RGD or SGDG motifs were altered.

 
Cell-cycle gene SuperArray showed that of the 7 genes expressed at high levels in DPSCs, P16, an inhibitor of CDK4, was down-regulated two-fold by Dentonin (Fig. 3Go). Among the genes expressed at low levels, ubiquitin protein ligase E3A (E6-AP) and human ubiquitin-related protein SUMO-1 mRNA (SUMO-1) were up-regulated greater than 7 times in the Dentonin group as compared with the control group.



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  		Figure 3. Cell-cycle SuperArray analysis showed that, of the more highly expressed genes, P16 was down-regulated approximately two-fold in the presence of Dentonin. Genes expressed at lower levels included E6-AP and SUMO-, which were up-regulated more than 7 times by Dentonin.

 
DPSC differentiation, as characterized by DSP secretion, showed no detectable difference between the control and Dentonin-treated cells (Fig. 1Go). Osteogenesis SuperArray showed strong expression of fibronectin and osteonectin mRNA by DPSCs in vitro, but there was no difference in the relative levels of expression of the osteogenesis-related genes, when mRNA from cells treated with Dentonin for 24 hrs was compared with that of controls. (See Fig. 4Go for 1 of 2 duplicate results.) Both the Dentonin and control cells formed nodules, and quantitative Alizarin Red staining showed no effect of Dentonin on mineralization (data not shown).



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  		Figure 4. Osteogenesis SuperArray showed that multiple osteogenesis-related genes were expressed by DPSCs in culture. Most highly expressed (black arrows) are fibronectin, osteonectin, decorin, CBFA1, and integrin ß1. There was no measurable difference between mRNA from DPSCs exposed to Dentonin in culture, as opposed to control (unexposed) cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Dentonin is a 23-mer peptide containing both RGD and SGDG motifs, and is located in the center of the full-length MEPE protein. The studies reported here show that Dentonin can enhance DPSC proliferation. Further analysis of the effect of Dentonin on cell-cycle-related genes showed a down-regulation of P16 in the presence of Dentonin. The maximal mitogenic activity of Dentonin required the presence of an intact RGD and SGDG, as shown by a reduction in activity when either of these motifs was altered.

The RGD motif is an integrin-binding unit. This binding is determined by the context and conformational structure around the RGD motif (Altroff et al., 2001; Han et al., 2003), and can trigger downstream signaling pathways to regulate cell adhesion, cell migration, and cell proliferation (Plow et al., 2000; Altroff et al., 2001). The variable results obtained with the RGD-altered Dentonin suggest that the integrin binding function of Dentonin enhances its ability to promote DPSC proliferation.

It is possible that the RGD-containing motif in Dentonin binds to certain integrin receptors and triggers a signaling pathway, which results in a down-regulation of P16. This down-regulation of P16 allows CDK4 to phosphorylate Rb. pRb then dissociates from the pRb/E2F complex, allowing E2F to activate the transcription of the target genes responsible for passing the G1/S restriction point to promote cell proliferation (Pollard and Earnshaw, 2002).

Dentonin-enhanced DPSC proliferation also required an intact SGDG motif. The SGDG motif may allow binding of glycosaminoglycans (GAGs) to inhibit degradation of the Dentonin peptide. This function may be similar to the effect of GAGs in protecting growth factors from proteolytic degradation (Syrokou et al., 1999). Protection of the intact peptide would allow the RGD domain of Dentonin to act as an integrin-binding site and promote cell proliferation. Thus, SGDG may act as a synergistic site for RGD to exert its function.

The up-regulation of E6-AP and SUMO-1, the ubiquitin-related proteins, in DPSCs by Dentonin may also contribute to the acceleration of cell-cycle progression. Both E6-AP and SUMO-1 may degrade or modify P53 (Franz et al., 2000; Baier et al., 2003), thus preventing cells from entering the P53 apoptotic pathway.

We were unable to detect any effect of Dentonin on DPSC differentiation by either Western blot, of DSP secretion, osteogenesis SuperArray, or mineralization assay. Therefore, Dentonin appears to have a specific effect on DPSC proliferation, but not on DPSC differentiation.

These results suggest a potential use of the 23-amino-acid peptide fragment of MEPE, named Dentonin or AC-100, in dentin repair. The vitality and the dentin repair capability of the pulp are dependent on odontoblast survival (Murray et al., 2000; Bleicher et al., 2001), with the total number of odontoblasts present an important factor in the pulp-dentin repair response to caries, trauma, and dental restoration (Murray et al., 2002). Therefore, it is possible that Dentonin may effectively stimulate DPSC proliferation in vivo, and consequently enhance the ability of injured pulp to survive trauma such as occurs in dental restorations. Further studies to determine the role of MEPE or Dentonin peptide in odontoblast formation will allow us to use these proteins to develop tools to promote the regeneration of dental tissues.


   ACKNOWLEDGMENTS
 
This investigation was supported by a Lee Hysan Scholarship and by USPHS Research Grant P01-DE09859 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Besthesda, MD, USA.

Received August 15, 2003; Last revision March 22, 2004; Accepted March 24, 2004


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