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Biological

Emdogain Stimulates Matrix Degradation by Osteoblasts

S. Goda1,3,*, H. Inoue2,3, Y. Kaneshita3, Y. Nagano3, Y. T. Ikeo1, J. Iida4 and N. Domae3

1 Departments of Biochemistry, Orthodontics,
2 Physiology, and
3 Internal Medicine, Osaka Dental University, 8-1 Kuzuha Hanazono-cho, Hirakata-shi, Osaka, 573-1121, Japan; and
4 Department of Laboratory Medicine and Pathology, Cancer Center, University of Minnesota, Minneapolis, USA

Correspondence: * corresponding author, goda{at}cc.osaka-dent.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Emdogain has been used clinically for periodontal regeneration, although the underlying molecular mechanisms are not clear at present. In this study, we hypothesized that Emdogain stimulated degradation of type I collagen via osteoblasts. We showed that Emdogain enhanced cell-mediated degradation of type I collagen in an MMP-dependent manner. Although MG-63 cells spontaneously produced a zymogen form of MMP-1, treatment with Emdogain significantly induced the generation of the active form of this enzyme. We demonstrated that MMP-3 was produced from MG63 cells in response to Emdogain in a MEK1/2-dependent manner. Concomitantly, blocking of MEK1/2 activation by U0126 significantly inhibited the generation of the active form of MMP-1 without affecting the total production of this collagenase. These results suggest that Emdogain facilitates tissue regeneration through the activation of the collagenase, MMP-1, that degrades matrix proteins in bone tissue microenvironments.

Key Words: Emdogain • matrix metalloproteinases • osteoblasts


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major objective of periodontal therapy is to stimulate regeneration of periodontal ligament (PDL) and alveolar bone. When applied to periodontal defects, Emdogain induces periodontal regeneration and improves therapeutic efficacy in concert with periodontal surgery (Gestrelius et al., 1997; Sculean et al., 2001; Cochran et al., 2003). Although previous studies showed that Emdogain promotes regeneration of PDL, root cementum, and alveolar bone in vivo (Hammarström et al., 1997; Heijl et al., 1997; Boyan et al., 2000), the mechanisms of its action still remain unclear.

Osteoblasts play a key role in remodeling bone by regulating matrix turnover, such as type I collagen. Matrix metalloproteinases (MMPs) are secreted by mesenchymal stromal lining cells and osteoblasts to generate the initiation sites for osteoclastic bone resorption at the beginning of the remodeling cycle (Sasaki et al., 2007). MMPs are endopeptidases that play a primary role in the degradation of extracellular matrix (ECM) proteins (Vu and Werb, 2000). Collagenases, including MMP-1, -8, -13, and -14, hydrolyze native collagens to generate 1/4 and 3/4 fragments, which are substrates for gelatinases and stromelysins (Visse and Nagase, 2003). Thus, it is hypothesized that the catalytic activity of collagenases initiates matrix turnovers in remodeling bone. Because Emdogain activated osteoblast cells to enhance mRNA expression of type I collagen, osteopontin, bone sialoprotein, and osteocalcin, it has potential therapeutic value as a material for the enhancement of bone remodeling (Yoneda et al., 2003).

Previous studies have shown that Emdogain regulates the production of MMP-1 and MMP-8 in gingival crevicular fluid (Okuda et al., 2001), although the functions of these collagenases are not clear. The purpose of this study was to characterize Emdogain for its ability to enhance matrix turnover and the production of MMPs, with the osteoblast cell line, MG-63.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
MG-63 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM supplemented with 10% heart-inactivated FBS, 2 mM glutamine, and 100 units/mL of penicillin/streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air.

Reagents and Antibodies
Anti-phospho-p44/42 (Thr202/Tyr204) and anti-p44/42 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Emdogain was obtained from Seikagakukogyo Corporation (Tokyo, Japan). U0126, MMP-1, and MMP-3 antibodies were purchased from Calbiochem (Darmstadt, Germany). DQ-collagen I was purchased from Molecular Probes (Eugene, OR, USA).

DQ-Collagen I Degradation Assay
Coverslips were coated with 25 µg/mL of quenched fluorescence substrate DQ-collagen I. MG-63 cells (5 x 104) were incubated with 100 µg/mL Emdogain for 20 hrs, followed by incubation on DQ-collagen I-coated plates for an additional 4 hrs. Cells were fixed with 2% paraformaldehyde and examined with 488-nm (excitation) and 533-nm (emission) fluorescence by confocal microscopy (Olympus LSM-GB200 confocal microscope, Olympus, Tokyo, Japan) with an oil immersion lens. Degradation of DQ-collagen I was visualized in an optical section as a green fluorescent signal. Differential interference contrast (DIC) was shown to visualize cells cultured on the matrix.

Western Blot Analysis
MG-63 (1 x 106) cells were incubated in serum-free medium with 50, 100, and 200 µg/mL of Emdogain for 24 hrs. The conditioned media were concentrated in Amicon Centriprep concentrators (MW cut-off, 10 kDa) (Millipore Corporation, Bedford, MA, USA) up to 10-fold to visualize proteins in Western blotting analysis. For studying phosphorylation of p44/42, we incubated MG-63 (1 x 106) cells in serum-free medium with 50, 100, or 200 µg/mL of Emdogain for the indicated periods described in the text. Samples were separated on 8% or 10% SDS polyacrylamide gels (SDS-PAGE) under reducing conditions. Proteins were electrophoretically transferred to Immobilon-P membranes and were incubated for 1 hr with primary antibodies in PBS containing 0.05% Tween-20 and 10% Blockace (Dainippon Pharm. Co., Tokyo, Japan). Peroxidase-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ, USA) was used at a 1:1000 dilution, and immunoreactive bands were visualized by means of Super Signal west pico chemiluminescent substrate (PIERCE Biotechnology Inc., Rockford, IL, USA). Signals on each membrane were analyzed with VersaDoc 5000 (BIO-RAD, Hercules, CA, USA).

Gelatin Zymography
The conditioning medium prepared as described above was subjected to gelatin zymography to visualize MMP-2 and MMP-9 as described previously (Goda et al., 2006). MG-63 (1 x 106) cells were incubated in serum-free DMEM with 50, 100, or 200 µg/mL of Emdogain for 24 hrs. The conditioning medium was resolved under non-reducing conditions on 10% SDS-PAGE impregnated with 1 mg/mL gelatin. Gels were rinsed once in 2.5% Triton X-100 for 30 min at room temperature and then incubated in a developing solution [50 mM Tris-HCl (pH 7.6), 5 mM CaCl2, 1 µM ZnCl2] for 8–16 hrs. Gels were stained with Coomassie blue and de-stained according to a standard protocol. Areas of gelatinolytic activity were detected as transparent bands. As a standard, a conditioning medium prepared from HT1080 cells stimulated with ConA was used to localize the pro-, intermediate, and active forms of MMP-2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Emdogain Stimulated the Degradation of Type I Collagen by MG-63
Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by osteoblasts play an essential role in bone remodeling and regeneration (Krane, 1995; Opdenakker et al., 2001). Previous clinical applications of Emdogain showed enhanced tissue regeneration, including bone formation (Heijl, 1997), suggesting that osteoblasts function to facilitate the process. We therefore utilized human osteoblasts, such as the MG-63 cell line, as a model system to evaluate Emdogain in the process of matrix turnover. We previously demonstrated that a quenched fluorescent derivative of type I collagen (DQ-collagen I) served as a substrate for MMPs for studying proteolysis by living cells (Goda et al., 2006). In this study, we used this assay system to examine the ability of MG-63 cells to degrade type I collagen. Degradation of DQ-collagen I was visualized in an optical section as green fluorescent signals. Although unstimulated MG-63 cells resulted in minimal signals of the degradation of type I collagen (Fig. 1a, 1cGo), Emdogain (100 µg/mL) enhanced the degradation of type I collagen visualized by the green fluorescence signals (Fig. 1d, 1fGo). Given the fact that type I collagen is a substrate for MMPs, we tested whether the tissue inhibitor of metalloproteinases (TIMP-2) would inhibit the proteolysis of type I collagen. TIMP-2 almost completely abrogated the proteolysis by the cells (Fig. 1g, 1iGo), indicating that MMPs plays a key role in the proteolysis of type I collagen.


Figure 1
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  		Figure 1. Emdogain enhanced MG-63 cells’ degradation of type I collagen. MG-63 cells were incubated in the presence (d, e, f) or absence (a, b, c) of 100 µg/mL Emdogain for 20 hrs and then incubated on glass coated with 25 µg/mL of quenched fluorescence substrate DQ-collagen I for an additional 4 hrs. TIMP-2 (20 µM) was incubated with Emdogain and MG-63 cells as described above (g, h, i). Degradation of type I collagen (green fluorescence) was detected by confocal microscopy (samples: excitation, 488 nm; emission, 530 nm). Pictures were taken at 40x magnification (a–i). Differential interference contrast (DIC) images are shown. These data are representative of more than 3 independent experiments.

 
Emdogain Enhanced the Generation of an Active Form of MMP-1 in MG-63 Cells
We then evaluated the production of collagenases (MMP-1, MMP-8, MMP-13, and MMP-14) from MG-63 cells stimulated by Emdogain. MG-63 cells spontaneously produced MMP-1 in the serum-free conditioned medium with 57 kDa (major) and 47 kDa (minor) molecular masses, which correspond to latent and active forms of this enzyme, respectively. Quantification of MMP-1 was performed densitometrically with NIH image software (Fig. 2AGo, upper). The intensities of each band are depicted as percent of maximum value. Importantly, when cells were cultured in the presence of Emdogain, the generation of the active form of MMP-1 was observed to occur in a dose-dependent manner (Fig. 2AGo). We separated the same samples (as in Fig. 2AGo) via gelatin zymography to test whether this enzyme was activated, since previous studies have shown that MMP-2 could act as a collagenase to some extent (Nagase and Woessner, 1999). While un-stimulated MG-63 cells spontaneously produced a latent form of MMP-2, neither the total amount of production nor the activation status of this enzyme was altered by stimulation with Emdogain (Fig. 2BGo), suggesting that the catalytic activity of MMP-2 is not involved in Emdogain-stimulated type I collagen degradation. Thus, the zymography shown in Fig. 2BGo served as a loading control for the Western blot illustrated in Fig. 2AGo. MMP-14 is a membrane-associated collagenase that promotes degradation of type I collagen and activates pro-MMP-2 (Seiki, 2003). However, MMP-14 was not activated in cell lysates in MG63 cells cultured in the presence of Emdogain at any concentration (not shown), consistent with the lack of activation of pro-MMP-2 (Fig. 2BGo). Emdogain did not affect the production of MMP-8 or MMP-13 (not shown). Given the fact that MMP-1 is a potent collagenase (Ohuchi et al., 1997), analysis of these data suggests that the active MMP-1 plays a key role in the proteolysis of type I collagen by Emdogain-stimulated MG-63 cells.


Figure 2
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  		Figure 2. Expression of MMPs from Emdogain-stimulated MG-63 cells. MG-63 cells (1 x 106 cells) were incubated in serum-free media containing 50, 100, and 200 µg/mL of Emdogain for 24 hrs and in the conditioned media as described in MATERIALS & METHODS. (A) The concentrated media were separated on 8% SDS-PAGE, blotted with anti-MMP-1 antibody, and visualized with a Super Signal west pico chemiluminescent substrate. Molecular markers (kDa) are shown in the left column. Quantification of MMP-1 (upper panel) was performed densitometrically with NIH image software. The intensities of each band are depicted as percent of maximum value (lower panel). (B) The same conditioned media were subjected to gelatinzymography as described in MATERIALS & METHODS. As a standard, conditioned medium prepared from HT1080 cells stimulated with ConA was used to localize inactive (upper) and active (lower) forms of MMP-2 and are shown as lines. These data are representative of more than 3 independent experiments.

 
Activation of MEK-ERK was Involved in the Degradation of Type I Collagen by Emdogain-stimulated MG-63 Cells
Previous studies showed that Emdogain stimulates MAP kinases in monocytic and periodontal ligament cells, inducing the formation of osteoclasts and a mitogenic response, respectively (Matsuda et al., 2002; Itoh et al., 2006). When MG-63 cells were cultured in the presence of 100 µg/mL Emdogain, ERK1/2 was phosphorylated in a time-dependent manner, with the maximum phosphorylation at 5 min (Fig. 3AGo, upper and lower panels). The total amount of ERK protein was not affected under the experimental conditions (Fig. 3AGo, middle panel). A synthetic specific inhibitor for ERK1/2, U0126, abolished the activation of this kinase in both unstimulated and Emdogain-stimulated MG-63 cells (Fig. 3BGo).


Figure 3
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  		Figure 3. Emdogain induced activation of ERK in MG-63 cells. (A) MG-63 cells were stimulated with 100 µg/mL EMD for the indicated times at 37°C. Cells were harvested, and lysates were resolved in 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was immunoblotted with anti-phospho-p44/42 antibody (upper panel) and then stripped and immunoblotted with anti-p44/42 antibody (middle panel). Molecular markers (kDa) are shown in the left column. (B) MG-63 cells were treated for 30 min with U0126 (5 and 10 µM) before stimulation with 100 µg/mL Emdogain for 5 min. Cells were harvested, and lysates were resolved in 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was immunoblotted with anti-phospho-p44/42 antibody (upper panel), and then stripped and immunoblotted with anti-p44/42 antibody (middle panel). Molecular markers (kDa) are shown in the left column. Quantification of phosphorylation of p44/42 was performed densitometrically and corrected to the amount of total p44/42 protein. The intensities of each band are depicted as percent of maximum value (lower panel). These data are representative of more than 3 independent experiments.

 
Previous studies have shown that the activation of ERK1/2 plays a key role in the induction of MMPs, including MMP-1 (Goda et al., 2006). We therefore tested whether this signaling pathway was involved in the proteolysis of type I collagen by Emdogain-stimulated MG-63 cells. Consistent with our results (Fig. 1Go), Emdogain enhanced proteolysis of type I collagen by MG-63 cells compared with unstimulated cells (Fig. 4AGo, d and f vs. a and c). Importantly, U0126 completely inhibited the proteolysis mediated by Emdogain-stimulated MG-63 cells (Fig. 4AGo, g and i). These results suggest that the activation of the ERK1/2 pathway as stimulated by Emdogain is involved in the production and/or activation of MMP-1.


Figure 4
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  		Figure 4. MEK-ERK pathways are important for Emdogain-stimulated MG-63 cell-mediated degradation of type I collagen. (A) MG-63 cells were pre-treated for 30 min with U0126 (10 µM) and then incubated in the presence or absence of 100 µg/mL Emdogain for 20 hrs. Cells were cultured on glass coated with 25 µg/mL of the quenched fluorescence substrate, DQ-collagen I. Degradation of type I collagen (green fluorescence) was detected by confocal microscopy (fluorescence: excitation, 488 nm; emission, 530 nm). Pictures were taken at 40x magnification (a–f). (B) The concentrated conditioned media prepared from unstimulated MG-63 cells (lane 1), U0126 (10 µM) (lane 2), Emdogain-stimulated MG-63 cells (lane 3), and Emdogain-stimulated MG-63 cells cultured in the presence of U0126 (10 µM) (lane 4) were separated on 8% SDS-PAGE. The membranes were blotted with anti-MMP-1 antibody and visualized with a Super Signal west pico chemiluminescent substrate. Molecular-weight markers (kDa) are shown in the left column. The same concentrated conditioned media were separated and transferred onto a membrane. (C) The membranes were blotted with anti-MMP-3 antibody and visualized with a Super Signal west pico chemiluminescent substrate. Molecular-weight markers (kDa) are shown in the left column. Quantification of MMP-1 (B) or MMP-3 (C) (upper panel) was performed densitometrically with NIH image software. The peak heights of each density are depicted as percent of maximum value (lower panel). These data are representative of more than 3 independent experiments.

 
U0126 significantly inhibited the activation of pro-MMP-1 in Emdogain-stimulated MG-63 cells, although the total amount of the collagenases was not altered (Fig. 4BGo). As a part of the study of the activation of pro-MMP-1, stromelysin-1 (MMP-3) has been reported as a direct activator through cleaving the prodomain of pro-MMP-1 between Gln80 and Phe81 (Nagase et al., 1992). Although MG-63 cells spontaneously produced MMP-3, Emdogain significantly enhanced MMP-3 production (Fig. 4CGo). Importantly, the enhanced production of MMP-3 reverted to the basal level in the presence of U0126 (Fig. 4CGo), suggesting that the activation of ERK1/2 is involved in the production of MMP-3 from Emdogain-stimulated MG-63 cells.

Taken together, these results suggest that Emdogain facilitates the proteolysis of type I collagen by MG-63 cells through the induction of MMP-3, which acts as an activator for pro-MMP-1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies demonstrated the indispensable role of cell-mediated "collagenolysis" in bone formation and growth of the skeleton (Holmbeck et al., 1999; Zhou et al., 2000). The involvement of matrix remodeling in bone has been studied by the elucidation of specific functional roles of MMPs, such as MMP-9 and MMP-14 (Vu et al., 1998; Holmbeck et al., 1999; Zhou et al., 2000). In this study, we demonstrated that Emdogain stimulated osteoblasts to degrade type I collagen in vitro through the catalytic activity of MMPs. Our results suggest that the activation of MMP-1 is one of the mechanisms for facilitating the degradation of type I collagen by Emdogain-stimulated MG-63 cells, which plays a key role in the processes of bone regeneration.

We demonstrated that MMP-1 was spontaneously produced from unstimulated MG-63 and was activated in the presence of Emdogain. Both degradation of type I collagen and the activation of pro-MMP-1 were positively correlated with the production of MMP-3 from Emdogain-stimulated MG-63 cells. Previous studies demonstrated that MMP-3 results in direct proteolysis of the Glu80-Phe81 bond in the prodomain of MMP-1, thus activating it (Nagase et al., 1992). Furthermore, MMP-1-mediated tumor invasion and matrix degradation required MMP-3 as an activator (Benbow et al., 1996). Thus, these results suggest that MMP-3 produced from Emdogain-stimulated MG-63 cells would act as an activator for pro-MMP-1. Previous studies showed that Emdogain decreased the production of MMP-1 in gingival crevicular fluid (Okuda et al., 2001). Although the in vivo function of Emdogain needs to be evaluated, it is possible that Emdogain may facilitate tissue and bone regeneration by regulating MMP-1 expression from distinct cell populations (i.e., neutrophils for tissue regeneration and osteoblasts for bone remodeling). Previous studies showed that Emdogain contains TGF-β and BMP-like activity (Suzuki et al., 2005). Furthermore, BMP-2 enhanced regeneration of bone and the production of MMPs (Wang et al., 1990; Takiguchi et al., 1998; Fujisaki et al., 2006). Although the details of the mechanisms of Emdogain for stimulating MMP-3 production from osteoblasts require further study, these results implicate soluble factors such as BMP-2 and TGF-β in Emdogain as potential activators for osteoblasts.

We have shown that the activation of ERK1/2 plays a key role in the induction of MMP-3 from Emdogain-stimulated MG-63 cells. Previous studies showed that activation of ERK1/2 plays a key role in the production of MMP-3 with various cell types and extracellular stimuli (Tanimura et al., 2003; Hoberg et al., 2007; Kajanne et al., 2007). Despite the fact that U0126 completely inhibited basal activation of ERK1/2 in MG-63 cells, the production of MMP-3 was approximately comparable with that in the non-treated control cells. These results suggest that spontaneous production of MMP-3 from MG-63 cells was independent of the activation of ERK1/2. Similarly, the production of MMP-1 from Emdogain-stimulated MG-63 cells was independent of the activation of ERK1/2. Furthermore, these results suggest that the production of MMPs is regulated via multiple signaling pathways stimulated by Emdogain in MG-63 cells. Although the characterization of the mechanisms of the production of MMP-3 from Emdogain-stimulated MG-63 cells is not clear at present, our results suggest that the induction of MMP-3 is one of the key steps for facilitating matrix turnover in bone environments.

In summary, our results suggest a model in which MMP-3 plays a role in bone regeneration by degrading matrix proteins and/or by activating a potent collagenase, MMP-1 in osteoblasts. In addition to ECM proteins, recent studies suggest that MMPs degrade various "non-classic" substrates in vivo (McCawley and Matrisian, 2001). For example, inflammatory chemokines such as monocyte chemoattractant proteins (MCPs) are a substrate for MMP-1 and MMP-3 and are inactivated by the cleavage at specific sites within the molecules (McQuibban et al., 2002). Thus, further studies will be needed to elucidate fully the mechanisms of Emdogain-stimulated tissue and bone formation by determining the substrates for MMPs, including MMP-1 and MMP-3, in tissues.


   ACKNOWLEDGMENTS
 
This work was supported by the High-Tech Research Center Project for Private University, a matching fund subsidy from MEXT, 2002–2006, and an Oral Implant Research Grant, Osaka Dental University (07-02).

Received for publication July 10, 2007. Revision received March 23, 2008. Accepted for publication May 19, 2008.


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 MATERIALS & METHODS
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 DISCUSSION
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Journal of Dental Research, Vol. 87, No. 8, 782-787 (2008)
DOI: 10.1177/154405910808700805
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