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Stem Cells Vol. 25 No. 1 January 2007, pp. 156 -164
doi:10.1634/stemcells.2006-0373; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

EphB/Ephrin-B Interaction Mediates Adult Stem Cell Attachment, Spreading, and Migration: Implications for Dental Tissue Repair

Agnieszka Stokowskia,b,c, Songtao Shid, Tao Sunc, Peter Mark Bartolde, Simon Andrea Koblara,b, Stan Gronthosc

aAustralian Research Council, Centre for the Molecular Genetics of Development,
bSchool of Molecular and Biomedical Science (Genetics), and
eColgate Australian Clinical Dental Research Centre, Dental School, University of Adelaide, Adelaide, South Australia, Australia;
cMesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science, Hanson Institute, Adelaide, South Australia, Australia;
dSchool of Dentistry, University of Southern California, Los Angeles, California, USA

Key Words. Eph • Ephrin • MSC • Dental pulp stem cell • Migration • Adhesion • Spreading

Correspondence: Stan Gronthos, B.Sc., M.Sc., Ph.D., Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, South Australia, Australia. Telephone: 61-8-8222-3460; Fax: 61-8-8222-3139; e-mail: stan.gronthos{at}imvs.sa.gov.au

Received June 20, 2006; accepted for publication September 20, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Human adult dental pulp stem cells (DPSCs) reside predominantly within the perivascular niche of dental pulp and are thought to originate from migrating neural crest cells during development. The Eph family of receptor tyrosine kinases and their ligands, the ephrin molecules, play an essential role in the migration of neural crest cells during development and stem cell niche maintenance. The present study examined the expression and function of the B-subclass Eph/ephrin molecules on DPSCs. Multiple receptors were primarily identified on DPSCs within the perivascular niche, whereas ephrin-B1 and ephrin-B3 were expressed by the surrounding pulp tissue. EphB/ephrin-B bidirectional signaling inhibited cell attachment and spreading, predominately via the mitogen-activated protein kinase (MAPK) pathway for forward signaling and phosphorylation of Src family tyrosine kinases via reverse ephrin-B signaling. DPSC migration was restricted through unidirectional ephrin-B1-activated EphB forward signaling, primarily signaling through the MAPK pathway. Furthermore, we observed that ephrin-B1 was downregulated in diseased adult teeth compared with paired uninjured controls. Collectively, these studies suggest that EphB/ephrin-B molecules play a role in restricting DPSC attachment and migration to maintain DPSCs within their stem cell niche under steady-state conditions. These results may have implications for dental pulp development and regeneration.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Tooth development occurs through the interaction between cranial neural crest-derived mesenchymal and epithelial cells [1]. We have recently identified a novel population of stem cells isolated from the dental pulp of human adult third molar teeth (dental pulp stem cells [DPSCs]) and exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth [SHED]) [2, 3]. DPSCs and SHED have been shown to possess self-renewal capacity, high proliferation potential, and the ability to undergo multilineage differentiation [3, 4]. These postnatal mesenchymal stem-like cells reside largely within specific locations within the tooth known as the perivascular niche [5] and are phenotypically similar to neural crest-derived pericytes that line the outer surfaces of blood vessels [6]. The tissue regeneration potential of human DPSCs and SHED has been demonstrated in vivo by their capacity to generate a dentine-pulp-like complex composed of mineralized matrix with tubules lined with odontoblasts and fibrous tissue containing blood vessels, similar to the arrangement of the dentine-pulp complex found in vivo [3, 4, 7].

A recent study has proposed that postnatal odontogenic precursor cells can be mobilized to sites of tooth repair following injury to the dentine matrix [8]. To date, little is known about the regulatory cues that restrict DPSCs within their stem cell niche or mediate their proliferation and mobilization to sites of tissue damage. Various transcription factors, growth factors, and matrix molecules are all thought to act as guidance cues to help orchestrate homeostasis and dynamic flux of different postnatal stem cell niches [9]. One well-known family of guidance molecules, the Eph receptor tyrosine kinase (RTK) family, has been implicated in neural stem cell migration along the subventricular zone and the proliferation of neural stem cells [10, 11]. Recent studies have also identified various Eph RTK family members during tooth development [12]. However, the expression of Eph/ephrin molecules in postnatal dental stem cell populations and their role in stem cell maintenance, proliferation, and migration have yet to be defined.

The Eph/ephrin family are contact-dependent molecules and well known for their role as mediating inhibitory or repulsive cellular responses. The family is divided into two subclasses based on structure for the ephrin ligands, where the A-subclass is glycosylphosphatidylinositol-tethered to the membrane, and the B-subclass is transmembrane. The receptors are divided into two subclasses characterized by the binding affinity for their cognate ligand, with promiscuous binding within subclasses but minimal interaction between subclasses, with the exception of EphA4 [13] and EphB2 [14].

Both subfamilies have been shown to signal through both the receptor (forward signaling) and the ligand (reverse signaling), mediating various responses depending on the mode of signaling. Many of the activated signaling pathways that predominantly bind conserved phosphorylated tyrosine kinases residing within the Eph receptor result in changes to cytoskeletal dynamics and adhesion. Activation of the kinase domain provides a docking site within the juxtamembrane domain for adaptor molecules that contain functional protein-interaction domains, such as Src homology 2 (SH2) and SH3 proteins. These adaptor proteins then mediate downstream signaling events [15] that result in functional changes such as altered cell shape, adhesion, motility, or proliferation [16, 17]. Furthermore, the suppression of the extracellular-signal related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway not only influences changes in the cytoskeleton but also inhibits proliferation and integrin-mediated cell adhesion [18, 19], whereas reverse signaling through ephrin-B ligands can be activated by either phosphorylation-dependent or phosphorylation-independent mechanisms. The activation by phosphorylation is similar to Eph forward signaling in that conserved tyrosine residues are phosphorylated within the cytoplasmic region [20–22]. The phosphorylation of conserved tyrosine residues induces an intracellular conformational change at the hairpin structure of the C-terminal region, subsequently exposing sites for adaptor protein binding [23], such as SH2 protein Grb4 [24]. The activation of ephrin-B reverse signaling through phosphorylation initiates a number of functional outcomes, including cytoskeletal changes [24].

Molecules of the Eph family are expressed by most tissue types during development and are important for maintenance of specific structures, such as rhombomere formation in the hindbrain, angiogenesis, neural stem cell proliferation, axon guidance, and migration of neural crest cells [17]. Eph/ephrin molecules are essential for correct cranial neural crest cells compartmentalization to specific rhombomeres in the hindbrain and migration during embryo development [25, 26]. The Eph receptors and ligands are expressed in complementary rhombomeres, restricting intermingling of cells at rhombomere boundaries by initiating a repulsive signal at the boundaries resulting in cell sorting between Eph- and ephrin-expressing cells [27, 28]. A response can be mediated either unidirectionally, through either Eph- or ephrin-expressing cells, or bidirectionally, where both Eph forward and ephrin reverse signaling occurs simultaneously through each cell [29, 30].

The present study examined the potential role of Eph/ephrin B-subclass interactions in regulating tissue maintenance and repair of postnatal dental tissue via the mobilization of DPSCs. We identified the expression of various Eph/ephrin B-subclass molecules by human DPSCs and within the pulp tissue and demonstrated that the bidirectional interaction between receptor and ligand restricted DPSCs to their niche under normal conditions, while permitting mobilization of DPSCs following injury through the downregulation of ephrin-B1.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Isolation of DPSCs
Normal impacted third molars were collected from adults (18–40 years of age) undergoing routine extractions at the Dental Clinic of the University of Adelaide. Informed consent was obtained in accordance with the guidelines set by the University of Adelaide and IMVS Human Subjects Research Committees. Tooth surfaces were cleaned and cracked open using a vise to reveal the pulp chamber. The pulp tissue was gently separated from the crown and root and then digested in a solution of 3 mg/ml collagenase type I (Worthington Biochem, Freehold, NJ, http://www.worthington-biochem.com) and 4 mg/ml dispase (Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com) for 1 hour at 37°C. Single-cell suspensions were obtained by passing the cells through a 70-µm strainer (Falcon; BD Labware, Franklin Lakes, NJ, http://www.bdbiosciences.com) and isolated as previously described. Cultures were established by seeding single-cell suspensions (1–2 x 105) of dental pulp into T-25 flasks (Corning Costar, Cambridge, MA, http://www.corning.com/lifesciences) in growth medium, {alpha}-modification of Eagle's medium (SAFC, Lenexa, KS, http://sigmaaldrich.com/SAFC/biosciences.html) supplemented with 20% fetal calf serum (SAFC), 100 µM L-ascorbic acid 2-phosphate (Wako Chemical, Tokyo, http://www.wako-chem.co.jp/english), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (SAFC), and then incubated at 37°C in 5% CO2.

Antibodies
Primary antibodies used were as follows: goat anti-human EphB1, goat anti-human EphB2, rabbit anti-human EphB4, rabbit anti-human ephrin-B1, and rabbit anti-human ephrin-B2, all 1:250; goat anti-human ephrin-B3, 1:100 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). Mouse monoclonal human vinculin (CBL 233), 1:20 (Cymbus Biotechnology, Chandlers Ford, Hampshire, U.K.); mouse anti-human STRO-1 and mouse IgM control (1A6.12), undiluted; CD146 (CC9.D3), 1:900; mouse IgG2a, control (1D4.5), 1:500 [5]; and rabbit-IgG and goat-IgG, 1:600 (Caltag, Burlingame, CA, http://www.caltag.com). Secondary antibodies were as follows: rabbit anti-goat biotin or goat anti-rabbit biotin, 1:50 (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com); goat anti-rabbit Alexa 488 and goat anti-mouse Alexa 488, 1:400 (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com); donkey anti-rabbit Cy3, 1:200; donkey anti-goat Cy3, 1:200 (Molecular Probes); and goat anti-mouse IgM-fluorescein isothiocyanate (FITC) and goat anti-mouse IgG2a-FITC, 1:50 (Caltag Laboratories). Tertiary antibodies were as follows: streptavidin Alexa 488, 1:500 (Molecular Probes); streptavidin-FITC, 1:100 (Caltag Laboratories); streptavidin-Texas Red, 1:100 (Caltag Laboratories).

Immunohistochemistry
Frozen sections (5 µm) of human pulp tissue were fixed with cold acetone at –20°C for 15 minutes and then washed in phosphate-buffered saline (PBS). The samples were blocked with either 5% nonimmune goat serum or rabbit serum for 1 hour at room temperature. Samples were incubated with 2 µg/ml primary antibodies (Eph/ephrin, STRO-1, CD146) overnight at 4°C. Mouse (IgM and IgG2a), goat, and rabbit (Ig) controls were treated under the same conditions. After being washed in PBS, the samples were incubated with the appropriate secondary antibodies (goat anti-rabbit biotin or rabbit anti-goat biotin) for 1 hour at room temperature. Dual-fluorescence labeling was achieved by adding secondary antibodies (goat anti-mouse IgM or FITC-conjugated IgG2a) and tertiary antibody (streptavidin-Texas Red) for 1 hour at room temperature. After being washed, the sections were coverslipped with fluorescence mountant.

Immunocytochemistry
Chamber slide or spreading assay cultures were fixed with 4% paraformaldehyde for 30 minutes at room temperature and then washed with PBS plus 0.1% Tween 20 (PBS-T). Cultures were blocked (10% horse serum in PBS-T and avidin-biotin blocking kit SP-2001; Vector Laboratories) for 30 minutes at room temperature and were then incubated with vinculin or mouse IgG in blocking solution overnight at 4°C. After being washed, mouse Alexa 488 and phalloidin-tetramethylrhodamine B isothiocyanate (TRITC), 1:2,000 (Sigma), were added in blocking solution for 2 hours at room temperature in the dark. Finally, cultures were washed and slides were coverslipped with 80% glycerol.

Spreading Assay
Fc-fusion proteins (human IgG-Fc 2.5 mg/ml, EphB2-Fc 200 µg/ml, and ephrin-B1-Fc 920 µg/ml) at 1, 5, and 10 µg/ml were preclustered with a 10-fold concentration of human anti-goat IgG (2.4 mg/ml) in PBS for 1 hour at room temperature. Ninety-six-well flat-bottom plates, chamber slides, or four-well plates were coated with poly(L-lysine) (0.01% solution; catalog no. P4707; Sigma-Aldrich) for 5 minutes at room temperature. Following the preclustering of the Fc, the samples were incubated in the coated wells for 2 hours at 37°C in a 5% CO2 incubator. The wells were washed in PBS to remove unbound -Fc. Cells were prepared as a single-cell suspension using PUCKS-EDTA (5 mM KCl, 130 mM NaCl, 3 mM NaHCO3, 5 mM D-glucose, 10 mM HEPES, pH 7.3, and 1 mM EDTA), washed in HHF (HEPES solution [Gibco, Grand Island, NY, http://www.invitrogen.com], 5% fetal calf serum [Equitech-Bio Inc., Kerrville, TX]), and resuspended in growth medium, and then 2 x 104 cells per well were added to each well and incubated for 3 hours at 37°C in 5% CO2. For the inhibitor assay, cells were incubated for 30 minutes at room temperature with signaling inhibitor U0126 (Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com), PP2 (gift from R. Ivell), LY294002 (gift from R. Ivell), or 0.1% dimethyl sulfoxide (DMSO) prior to being added to the well. The cultures were then fixed and stained with phalloidin-TRITC and/or vinculin as described above. For consistency, images of the cultures were taken in the center of each well.

Real-Time Polymerase Chain Reaction
RNA was extracted from normal and injured pulp tissue from human donors. Briefly, total RNA was extracted from pulp tissue using a modified protocol combining the TRIzol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) method with the RNeasy extraction system RNA cleanup protocol (Qiagen, Hilden, Germany, http://www1.qiagen.com). RNA samples were quantified by spectrophotometer (Eppendorf, Hamburg, Germany, http://www.eppendorf.com), and RNA integrity was checked on 1% agarose gels using a deionized formamide-based loading buffer. Reverse transcription reactions were performed using Superscript III reverse transcriptase (Invitrogen). cDNA samples were diluted to a uniform concentration of 50 ng/µl. Real-time polymerase chain reactions (PCRs) were performed using TaqMan master mix on an ABI SDS 7000 light cycler driven by ABI prism SDS v1.1 (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com). TaqMan primers were designed using Primer Express v2.0 (Applied BioSystems) and synthesized locally (GeneWorks, Hindmarsh, SA, Australia, http://www.geneworks.com.au/). The following primers were used at a final concentration of 300 nM: TATA box binding protein (TBP) forward (CTG GAA AAG TTG TAT TAA CAG GTG CT), reverse (CCA TCA CGC CAC AGT TTC C); EphB1 (AF037331) forward (GTG GCT ACG ATG AAA ACC TGA AC), reverse (CTG GTT GGG CTC GAA GAC AT); EphB2 (AF025304) forward (ATG AAC ACG ATC CGC ACG TA), reverse (TTG GTC CGT AGC CAG TTG TTC T); EphB3 (NM_004443) forward (TGT GTA ATG TGC GCG AGT CA), reverse (TTG CAG TCA CGC ACA GTG AA); EphB4 (NM_004444) forward (GCC GCA GCT TTG GAA GAG), reverse (CAT CCA GGC CGC TCA GTT); ephrin-B1 (NM_004429) forward (AGC TCC CTC AAC CCC AAG TT), reverse (GGC AGA TGA TGT CCA GCT TGT); ephrin-B2 (NM_004093) forward (CCT CTC CTC AAC TGT GCC AAA), reverse (CCC AGA GGT TAG GGC TGA ATT); ephrin-B3 (NM_001406) forward (TGT CTA CTG GAA CTC GGC GAA T), and reverse (TCC CCG ATC TGA GGG TAC AG). Reactions for each sample were performed in either triplicate or quadruplicate.

Migration Assay
Fc-fusion proteins (2.5 mg/ml human IgG-Fc, 200 µg/ml EphB2-Fc, and 920 µg/ml ephrin-B1-Fc) at 10 µg/ml were preclustered with a 10-fold concentration of human anti-goat IgG (2.4 mg/ml) in PBS for 1 hour at room temperature. Cells were prepared as a single-cell suspension using PUCKS-EDTA, washed in HHF, and resuspended in growth medium, and then 3 x 104 cells per well were added to the top chamber of each transwell (Corning Costar) and incubated for 24 hours at 37°C in 5% CO2. For the inhibitor assay, cells were incubated for 30 minutes at room temperature with signaling inhibitor 10 µM U0126 (New England Biolabs, Ipswich, MA, http://www.neb.com/) or 0.1% DMSO prior to being added to the transwell. The cultures were then fixed, the inside of the transwell was cleaned using a cotton bud to remove any cells that had not migrated through the membrane, and cells were then stained with 4,6-diamidino-2-phenylindole to visualize the nucleus. The assay was repeated in duplicate wells, and images were taken at three random positions within the well for each well. The number of nuclei were counted in each image and represented as the average cell count/field of view.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Distribution of EphB/Ephrin-B Molecules Within Human Dental Pulp Tissue In Vivo
Previous studies have demonstrated that STRO-1-positive DPSCs reside in a perivascular niche within adult human dental pulp tissue [5]. In situ immunohistochemical staining of frozen human dental pulp sections (Fig. 1) revealed that EphB1, ephrin-B1, and ephrin-B3 were strongly expressed by cells of the mature odontoblast layer and the fibrous tissue, surrounding STRO-1-positive perivascular cells within the pulp tissue. EphB4 and ephrin-B2 were expressed predominantly on vascular structures, away from the differentiated odontoblast layer, as demonstrated by dual-color costaining with the STRO-1 (Fig. 1B, 1C, 1E) and CD146 antigens (Fig. 1G, 1H), respectively. Similar expression patterns were observed for EphB2, albeit at lower levels. EphB4 expression appeared as punctate staining throughout the whole cell surrounded by either STRO-1 or CD146 staining, suggesting that endothelial cells also expressed EphB4. Likewise, ephrin-B2 appeared as punctate staining within the perivasculature, localizing with STRO-1-expressing cells along the outer perimeter of the blood vessel wall, and with CD146 at the inner edge of the blood vessel wall lined with endothelial cells.


Figure 1
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  		Figure 1. EphB/ephrin-B molecules are expressed in adult human third molar pulp tissue. Human third molar pulp tissue (5-µm frozen sections) was costained with B-subclass Eph receptor or ephrin ligand (Texas Red) and the mesenchymal stem cell marker STRO-1 (fluorescein isothiocyanate [FITC]) (A–F) or the perivascular marker CD146 (FITC) (G, H). EphB1 (A), EphB2 (B), EphB4 (C), and ephrin-B2 (E) colocalized with STRO-1, whereas EphB1 (A), in addition to ephrin-B1 (D) and ephrin-B3 (F), was strongly expressed by the surrounding fibrous pulp tissue (arrows). (G–H): EphB4 (G) and ephrin-B2 (H) were also identified in the perivasculature. Scale bar = 20 µm. (I): Gene expression profile studies identified several Eph/ephrin of B-subclass transcripts expressed by ex vivo-expanded human dental pulp stem cells. Specifically, we confirmed the mRNA expression of EphB1, B2, B3, B4, B6, and ephrin-B1-B3 using real-time polymerase chain reaction (PCR). The lower the cycle threshold, the higher the gene expression. TBP was the positive housekeeping gene control for reverse transcription-PCR. Abbreviation: TBP, TATA box binding protein.

 
Eph/Ephrin Interaction Inhibits DPSC Attachment and Spreading
Gene expression profile studies confirmed the expression of different Eph/ephrin-B-subclass transcripts by ex vivo-expanded human DPSCs, including EphB1, B2, B3, B4, B6, and ephrin-B1-B3 using real-time PCR (Fig. 1I). We next used an established spreading assay to investigate the role of B-class Eph/ephrin molecules in mediating the maintenance of STRO-1-positive DPSCs within their perivascular niche. Secondary cultures of human DPSCs exhibited a significantly rounder and smaller (slightly less than half the size) morphology when exposed to optimal concentrations (10 µg/ml) of either EphB2-Fc (Student's t-test, p < .0046) or ephrin-B1-Fc (Student's t-test, p < .01), in comparison with the human IgG-Fc controls (Fig. 2). Exposure of DPSCs to either EphB4-Fc or ephrin-B3-Fc failed to stimulate the same response (data not shown). On closer examination, cells treated with EphB2-Fc and ephrin-B1-Fc formed tight rings of actin at the edge of the cell, reminiscent of blebbing [31]. In contrast, control cells exposed to human IgG-Fc showed well-splayed actin with extending filopodia and lamellipodia (Fig. 2A–2I). These data suggested that B-subclass receptors and ligands contributed to the attachment, adhesion, and, consequently, spreading of DPSCs in vitro.


Figure 2
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  		Figure 2. Dental pulp stem cells (DPSCs) undergo morphological changes when exposed to EphB2-Fc or ephrin-B1-Fc. Single-cell suspensions of cultured DPSCs were used for spreading assays and then stained with phalloidin-tetramethylrhodamine B isothiocyanate. Different DPSC cultures, NHT 6-99 (A, D, G), NHT 5-01 (B, E, H), and NHT 7-99 (C, F, I), demonstrated a rounding response in the presence of 10 µg/ml EphB2-Fc (D–F) or ephrin-B1-Fc (G–I), but not human-IgG-Fc (A–C). The roundness (J) and surface area (K) of DPSCs derived from three independent donors was analyzed with Scion Image software. In all three donors, DPSCs were significantly rounder and smaller when exposed to EphB2-Fc or ephrin-B1-Fc compared with human IgG-Fc. *, p < .036; #, p < .0046; {bigwedge}, p < .01. Scale bar = 20 µm. Abbreviation: NHT, normal human teeth.

 
During this analysis, we observed that fewer DPSCs remained attached in the presence of EphB2-Fc or ephrin-B1-Fc compared with human IgG-Fc. Time-lapse imaging indicated that the majority of DPSCs remained rounded, continuously slightly extending and subsequently retracting their filopodia over the 3-hour period that they were exposed to either 10 µg/ml EphB2-Fc or ephrin-B1-Fc. (Fig. 3). Conversely, DPSCs attached and spread substantially during the 3-hour incubation in the presence of human IgG-Fc. These data implied that the lack of attachment, adhesion, and spreading was in response to either EphB2 or ephrin-B1 interaction with their reciprocal ligand or receptor expressed by DPSCs and not the -Fc itself. Importantly, the reduction in cell attachment was not a consequence of early or late stage apoptosis, as confirmed by the absence of annexin V and trypan blue staining in bound and unbound cells, respectively (data not shown). Collectively, these results imply that B-subclass Eph/ephrin interactions may act to disrupt focal adhesions, which are essential for DPSC attachment and spreading.


Figure 3
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  		Figure 3. EphB2-Fc or ephrin-B1-Fc inhibition of dental pulp stem cell (DPSC) adhesion and spreading. Representative time-lapse video micrographs of still images at hourly time points indicated that when DPSCs were in contact with human IgG-Fc (A), they attached and spread their processes (arrowhead), forming connections with surrounding cells. When DPSCs were in contact with EphB2-Fc (B) or ephrin-B1-Fc (C), cells that attached were restricted in spreading their cytoplasmic processes (arrowhead). Scale bar = 20 µm.

 
To further examine the underlying mechanism of attachment of DPSCs in response to EphB/ephrin-B interactions, spreading assays were performed as described above and then co-immunostained for both vinculin and F-actin to examine the distribution of focal adhesion complexes and cytoskeletal morphology, respectively. The distribution of vinculin on DPSCs exposed to human IgG-Fc (Fig. 4A) appeared throughout and at the leading edge of the cell as diffuse staining, characteristic of normal attachment and spreading. In contrast, vinculin distribution appeared as circular patches, surrounded by rings of F-actin in the absence of ECM molecules following contact with EphB2-Fc (Fig. 4B) or ephrin-B1-Fc (Fig. 4C). The aberrant dissemination of vinculin coincided with the appearance of spreading initiation centers (SICs), which are present only at early stages of cell spreading, as previously described by de Hoog et al. [32]. Three-dimensional confocal imaging using z-serial analysis confirmed that F-actin distribution of DPSCs exposed to EphB2-Fc (Fig. 4B') or ephrin-B1-Fc (Fig. 4C') formed ring structures surrounding vinculin (Fig. 4B''–4C'') or the SIC [32] and was not actually part of the SIC itself.


Figure 4
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  		Figure 4. Mature focal adhesion complexes do not form in dental pulp stem cells (DPSCs) when exposed to EphB2-Fc or ephrin-B1-Fc. DPSCs were treated with 10 µg/ml of human IgG-Fc (A), EphB2-Fc (D), or ephrin-B1-Fc (G). Cells were subsequently stained with phalloidin-TRITC (red) and anti-vinculin (green), indicating F-actin distribution and focal adhesion complexes, respectively. The same cell was visualized using confocal stacks of 1.5-µm slices, demonstrating that vinculin remained as circular patches surrounded by F-actin when exposed to EphB2-Fc or ephrin-B1-Fc. Individual slices down through the cell (1.5 µm, left to right) for F-actin (B'–C') and vinculin (B''–C'') distribution are illustrated. Scale bar = 20 µm (A), 20 µm (B, C).

 
DPSC Migration Is Mediated by Eph/Ephrin Interactions
A transwell migration assay was used to assess the effect of B-class Eph/ephrin interactions during DPSC migration under normal growth conditions. This established assay has previously been used to investigate endothelial cell migration in response to EphB/ephrin-B signaling [33]. In the present study, DPSCs were seeded into the top well, separated by an 8-µm pore filter in the presence of either B-class Eph-Fc, ephrin-Fc, or human IgG-Fc added to the bottom chamber. The data showed that DPSCs were capable of migrating freely through the filter and adhering to the underside in the presence of human IgG-Fc (Fig. 5). Surprisingly, DPSC migration was not inhibited by EphB2-Fc, in contrast to the observed inhibitory effects of EphB2-Fc on DPSC attachment and spreading. However, when DPSCs were exposed to ephrin-B1-Fc, there was a significant (p < .0097) reduction in their migration potential compared with those wells containing human IgG-Fc (Fig. 5).


Figure 5
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  		Figure 5. EphB forward signaling inhibits dental pulp stem cell (DPSC) migration in vitro. DPSCs plated in the top chamber of a transwell migration assay migrated through the 8-µm pore membrane to the bottom chamber following a 24-hour incubation. Cells remaining in the top chamber were removed, whereas the cells that had migrated through were fixed and stained with 4,6-diamidino-2-phenylindole to visualize the nucleus. Images were taken in several random regions of the membrane, and the number of nuclei was counted. The graph represents the average of six independent cell counts and three fields of view from duplicate membranes. Error bars = SEM. DPSCs were exposed to 10 µg/ml human IgG-Fc, and EphB2-Fc migrated through the membrane, whereas significantly fewer cells migrated in the presence of ephrin-B1-Fc. *, p < .0097. Scale bar = 100 µm. Abbreviation: av, average.

 
Eph/Ephrin Signaling Is Required for DPSC Attachment, Spreading, and Migration
Specific inhibitors of Eph/ephrin family of RTK signaling cascades were added to the spreading assays to identify the signaling pathways involved in Eph/ephrin-mediated DPSC attachment and spreading. When DPSCs were preincubated with the mitogen-activated protein kinase kinase (MEK)/ERK inhibitor U0126 [34], which blocks the MAPK pathway, DPSC morphology reverted back to that of the control cells (Fig. 6A). U0126 was found to increase attachment and spreading of human DPSCs in a dose-dependent manner in the presence of ephrin-B1-Fc, at an optimal concentration of 10 µM U0126 (data not shown). In contrast, neither the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 [35] nor the Src family kinase inhibitor PP2 [36] influenced the attachment or spreading of human DPSCs to any significant degree in response to ephrin-B1-Fc (Fig. 6A). Furthermore, no change in DPSC morphology was observed in the presence of either U0126 or LY294002 (Fig. 6A) when in contact with EphB2-Fc. However, when human DPSCs were incubated with 10 µM PP2 prior to their exposure to EphB2-Fc, more cells attached and spread on the EphB2-Fc surface than the control cells in the absence of PP2 (Fig. 6A). The PP2 inhibitor functions by preventing the phosphorylation of Src family kinases. The cytoplasmic region of ephrin-B molecules contains an SH2 domain that spans three of five conserved tyrosine kinase residues. Upon tyrosine phosphorylation, ephrin-B reverse signaling predominantly mediates a response through this SH2 domain, which develops high-affinity binding for SH2-containing proteins, such as Grb4 [24]. The findings from this study demonstrate that although the restricted attachment and spreading of human DPSCs was mainly mediated through MAPK pathway for Eph forward signaling, cell rounding and detachment were also found to be mediated, in part, through tyrosine phosphorylation-dependent ephrin-B reverse signaling, most probably via an SH2-containing protein.


Figure 6
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  		Figure 6. Eph/ephrin signaling is required for dental pulp stem cell (DPSC) attachment, spreading and migration. (A): EphB receptor predominantly mediated forward signaling through the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. Representative images of cells preincubated with 0.1% DMSO in growth medium did not change their previously observed morphology in response to either human IgG-Fc, EphB2-Fc or ephrin-B1-Fc. Inhibition of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways using inhibitors U0126 and LY294002 did not alter the morphology of DPSCs in response to EphB2-Fc. DPSCs mediated ephrin-B reverse signaling in a phosphorylation-dependent manner through the SH2 domains. DPSCs preincubated with PP2 did not round or detach in response to the EphB2-Fc, instead displaying a spread morphology, as observed with the human IgG-Fc. DPSCs preincubated with either LY294002 or PP2, when in contact with ephrin-B1-Fc, did not change the rounding response of DPSCs mediated through Eph forward signaling. Alternatively, U0126-preincubated DPSCs no longer rounded and detached in response to ephrin-B1-Fc, instead displaying normal spreading morphology, as observed with the human IgG-Fc. (B): DPSCs exposed to the transwell migration assay were treated with either 0.1% DMSO or 10 µM U0126 prior to their exposure to either 10 µg/ml human IgG-Fc or ephrin-B1-Fc. DPSCs exposed to human IgG-Fc migrated through the 8-µm pore membrane, whereas DPSCs in contact with ephrin-B1-Fc reduced their migration significantly compared with human IgG-Fc (#, p < .00001). However, in the presence of MEK/ERK inhibitor U0126, DPSCs were no longer responsive to ephrin-B1 interaction, significantly (p < .0003) increasing their migration through the membrane. Scale bar = 20 µm. Abbreviations: av, average; DMSO, dimethyl sulfoxide.

 
In parallel studies, DPSCs were treated with the MAPK pathway inhibitor U0126 to examine the migration potential following blocking of Eph forward signaling (Fig. 6B). The data showed that significantly (p < .0003) more DPSCs migrated through the filter in the presence of ephrin-B1-Fc when preincubated with the inhibitor U0126 when in contact with ephrin-B1-Fc, in comparison with DPSCs in contact with ephrin-B1-Fc alone. Although the MAPK signaling pathway appeared to be the major constituent involved in mediating the inhibitory response, the number of cells that were able to migrate through the membrane with U0126 was still lower than in the presence of human IgG-Fc control, suggesting that there might be other signaling pathways involved during EphB forward signaling.

ephrin-B1 Is Downregulated in Dental Pulp Following Injury
We next examined the EphB/ephrin-B expression in freshly isolated dental pulp tissue under normal steady-state conditions and following tooth injury. Total RNA was isolated from collagenase- and dispase-digested pulp tissue derived from normal and caries-affected third molars isolated from patients undergoing routine extractions. Real-time PCR was subsequently performed to compare EphB and ephrin-B gene expression between normal and injured samples from three independent donors. The results demonstrated no significant difference in EphB1, EphB2, EphB4, ephrin-B2, and ephrin-B3 gene expression between normal and injured pulp tissue from the same individuals (Fig. 7). However, a significant (p < .012) 1.8-fold decrease in ephrin-B1 expression was observed between paired normal and caries-affected molars isolated from three subjects.


Figure 7
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  		Figure 7. Ephrin-B1 gene expression was significantly reduced following tooth injury. Donors provided two molars, one normal and one diseased. The pulp was removed from the samples, and mRNA was isolated. Samples were isolated from three donors (n = 3). Real-time polymerase chain reaction comparing EphB (A) and ephrin-B (B) gene expression between normal and injured samples, represented as the difference in cycle threshold relative to control gene TATA box binding protein, identified no significant difference in EphB gene expression, whereas a significant (#, p < .012) 1.8-fold decrease in ephrin-B1 expression was observed following injury.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
We have previously demonstrated that human DPSCs have the capacity to regenerate organized tooth-like structures when transferred subcutaneously into immunocompromised mice [3, 4, 7]. More recently, Tecles et al. confirmed that odontogenic precursor cells could be mobilized from their perivascular niche to sites of injured pulp or dentine tissue [8]. However, the mechanisms responsible for the recruitment and differentiation of DPSCs have yet to be defined. It has been shown previously that following spinal cord injury, EphB/ephrin-B molecules initially downregulate their expression, whereas cells migrate and infiltrate the injury site and then upregulate once migration has ceased [37]. Furthermore, Eph/ephrin molecules of both subclasses have been implicated in stem cell proliferation responses [10, 11]. The present study suggests that the B-subclass Eph/ephrin molecules are critical factors required for DPSC niche maintenance under steady-state conditions and DPSC recruitment during tissue repair.

Eph/ephrin interactions have been shown to restrict the intermingling of different populations of embryonic neural precursor cells from developing hindbrain rhombomeres [28, 38, 39]. Therefore, we hypothesized that Eph/ephrin molecules may act to confine DPSCs within their perivascular niche by restricting their intermingling between different cell populations via the prevention of focal adhesion formation. The spreading assay analysis and time-lapse imaging data suggested that DPSCs were inhibited from spreading, where they remained in a rounded state in the presence of either the EphB receptor or ephrin-B ligand. However, rather than losing focal adhesions, as previously shown during boundary formation, DPSCs were found to be limited in their ability to form mature focal adhesions. Our data are in accord with studies showing neural crest cell loss of focal adhesion complexes in response to ephrin-B1-Fc [40].

Focal adhesion complexes comprise a multitude of proteins that together are essential for cell attachment/adhesion, spreading, and migration [41, 42]. Vinculin, a major component of focal adhesion complexes, specifically attaches the actin cytoskeleton [43] to various cell adhesion molecules/receptors [42] associated with the process of cell adhesion, spreading, and migration. Interestingly, vinculin distribution appeared as circular patches on DPSCs exposed to EphB2-Fc or ephrin-B1-Fc but not to the same extent with human IgG-Fc following the spreading assay. These circular patches appeared similar to the SICs [32], or dot-like adhesions [44], which are required for early stages of cell spreading, prior to the maturation of focal adhesions. In the present study, three-dimensional confocal microscopy imaging of double-labeled F-actin and vinculin by DPSCs treated with either EphB2-Fc or ephrin-B1-Fc revealed that the blebbing of actin was not caused by cell death but by the formation of actin ring structures, possibly ensheathing vinculin-expressing SICs. Time-lapse imaging further demonstrated that DPSCs were inhibited from spreading on either EphB2-Fc or ephrin-B1-Fc, but not on human IgG-Fc, where DPSCs continuously tried to extend their processes but were retarded in doing so, inhibiting the maturation of focal adhesion complexes and thus the movement of DPSCs.

The constant movement of the DPSC filopodia and lamellipodia in response to EphB/ephrin-B interaction suggests active signaling in both the EphB- and ephrin-B-expressing cells. Our results suggest that DPSCs signal bidirectionally through both the Eph- and ephrin-expressing DPSCs to restrict cell intermingling. This concept is supported by Wilkinson et al., who showed that bidirectional signaling is required for restricted cell intermingling [29]. However, in vitro migration analysis suggested that DPSC mobilization only required unidirectional signaling via EphB-expressing DPSCs in response to contact with ephrin-B1-Fc. In situ studies of human teeth demonstrated that EphB-expressing, STRO-1-positive DPSCs situated within their perivascular niche were surrounded by high-ephrin-B-expressing pulp tissue. However, we observed a significant downregulation of ephrin-B1 expression in damaged teeth compared with normal samples taken from the same individuals. Hence, we suggest these data support the concept that there is dynamic EphB/ephrin-B bidirectional signaling that maintains DPSCs within their niche by limiting the intermingling within the surrounding environment. However, DPSCs might potentially migrate to the injury site when EphB unidirectional signaling is interrupted following damage to the dentin and pulp tissues.

Previous studies have indicated that EphB-expressing cells retract their processes and round through endocytosis [45], whereas rounding and detachment of ephrin-B-expressing cells required tyrosine phosphorylation [24]. Our studies indicated that restricted DPSC attachment, spreading, and migration were all predominantly mediated through the ERK/MAPK pathway, which is important not only for cytoskeletal reorganization but also for cell division. The de-adhesion and rounding response of DPSCs was mostly reverted in the presence of MEK/ERK inhibitor, suggesting that the forward signaling cascade was primarily mediated through the MAPK pathway and not via PI3K signaling. Although the MAPK pathway was shown to be essential for DPSC rounding and detachment, we are unsure whether this response is mediated through RasGAP or R-Ras. It is plausible that both pathways are required for alternative events, such that the RasGAP-mediated downregulation of MAPK may be required for DPSC rounding, as shown for neurite retraction [18], whereas R-Ras, which inhibits integrin activation of MAPK, may act to regulate DPSC adhesion.

Reverse signaling of ephrin-B molecules can be mediated in a phosphotyrosine-dependent or -independent manner [20, 46, 47]. We observed that ephrin-B-expressing DPSCs mediate their signal through a phosphotyrosine-dependent pathway via Src homology 2/3 (SH2/SH3) domains. We postulate that ephrin-B1 reverse phosphorylation-dependent signaling may be mediated by SH2/SH3 adaptor protein Grb4, where Grb4 is able to recruit CAP (Cbl-associated protein), resulting in loss of stress fibers via the lack of F-actin, the disassembly of focal adhesions, and consequently cell rounding via ephrin-B1 reverse signaling [24]. Interestingly, when the phosphorylation of Src family kinases was blocked using PP2, where Src family kinases predominantly mediate their signal through proteins containing SH2 domains, there appeared to be more Eph-expressing DPSCs remaining attached following the spreading assay. However, this did not occur to the extent observed with the MAPK inhibitor, suggesting that SH2 binding proteins may not be essential for DPSC rounding and cell adhesion.

The present study demonstrated that DPSC migration was inhibited through Eph forward signaling. This inhibitory response could be partially reversed by the addition of a MAPK inhibitor, suggesting that at least for DPSC migration, other signaling pathways may also mediate cellular migration of DPSCs. For example, the PI3K signaling pathway is necessary for endothelial cells migration and proliferation through EphB receptors [33, 48, 49]. Although it was surprising that the PI3K signaling pathway was not essential for DPSC attachment and spreading, it might be required in combination with MAPK signaling for DPSC migration [50]. In addition, almost half as many DPSCs appeared to migrate in response to ephrin-B1 in comparison with the human IgG-Fc controls. However, there were a number of cells that were not responsive to ephrin-B1 interaction, suggesting that the inhibitory response could be mediated through ephrin-B3, also present on pulp tissue surrounding the DPSC niche.

Summary
The findings from this study present a model to understand the molecular mechanism underlying the maintenance and recruitment of a mesenchymal stem cell-like population in human teeth. We have shown that Eph/ephrin interactions may contribute to the localization of DPSCs within adult human teeth and the maintenance of DPSCs within their niche. The subsequent mobilization of DPSCs following injury to the dentine surfaces within the adult dental pulp tissue may be mediated by Eph/ephrin interactions and explain the expression of this family in human adult teeth before and after injury. If tissue engineering were to be used to repair adult human teeth, the manipulation of molecules such as the Eph/ephrin family would be worthy of future in vivo experimentation.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We thank Dr. Warren Flood for assistance with real-time reverse transcription-PCR setup, Dr. Gafar Sarvestani for assistance with confocal microscope imaging (DETMOLD Imaging Core Facility, Hanson Institute, Adelaide, SA, Australia), Dr. Ravinder Anand-Ivell for useful discussion on signaling pathways and gifts of signaling pathway inhibitors. Thanks also go to the Koblar and Gronthos laboratory members and to Anne Hamilton-Bruce and Craig Arthur for helpful discussions. This work was supported in part by Australian Research Council Special Research Centre for the Molecular Genetics of Development Grant S00001531, a University of Adelaide Faculty of Science Postgraduate Scholarship, an ARC Special Research Centre for the Molecular Genetics of Development Postgraduate Scholarship (to A.S.), and National Health & Medical Research Council Project Grant 242804. S.A.K. and S.G. are joint senior authors.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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