Background
Two major objectives of periodontal therapy are regenerating the periodontal ligament (PDL) and rebuilding alveolar bone lost as a result of periodontal disease. Previous experimental models and clinical studies have shown that enamel matrix-derived (EMD) protein promotes generation of PDL, root cementum and alveolar bone[
1‐
3]. EMD protein also activates osteoblasts cells in vitro, leading to a wound-healing response[
4] and generation of alkaline phosphatase[
5]. In addition, EMD protein regulates the production of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in gingival crevicular fluid[
6,
7].
Bone is continuously remodeled, and the amount of new bone depends on the balance between bone formation and resorption, which are mediated by osteoblasts, osteoclasts and osteocytes. Disturbed extracellular matrix (ECM) turnover leads to bone loss and its associated diseases, such as periodontitis. Osteoblasts are bone-remodeling cells that differentiate from mesenchymal stem cells and secrete ECM protein, which is subsequently mineralized by osteoblasts. MMPs are zinc atom-dependent endopeptidases that play a primary role in the degradation of ECM proteins[
8]. Osteoblasts and osteocytes also produce MMPs such as MMP-2 and MMP-13[
7,
9]. The function of MMP-2 is to degrade ECM proteins and promote remodeling and regeneration of bone tissue[
10].
Mitogen-activated protein kinases (MAPKs) are important signal transducing enzymes involved in cellular regulation. Recent studies using a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor showed that cytokine stimulation of MMP-2 synthesis is involved in p38 MAPK signaling[
11,
12].
The purpose of this study was to clarify the effects of EMD protein on the production and activation of MMP-2 using an osteoblast-like cell line, that is, MG-63. We found that EMD protein promoted the degradation of gelatin on MG-63 cells and enhanced the activation of MMP-2 in MG-63 cells. The EMD protein signaling pathways depends on p38 MAPK. These results suggest that selective regulation of MMP-2 production and subsequent activation of MMP-2 by EMD protein in MG-63 cells leads to remodeling and regeneration of periodontal connective tissue.
Methods
Cell line
Osteoblasts (MG-63 cell line; American Type Culture Collection, Rockville, MA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS (Equitech-Bio Inc., TX, USA), 2 mM glutamine and 100 units/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% CO2 in air.
DQ gelatin degradation assay
Coverslips were coated with 100 μg/ml quenched fluorescence substrate DQ-gelatin (Molecular Probes, Eugene, OR). MG-63 cells were incubated with 100 μg/ml EMD protein (Seikagaku-kogyo Corp., Osaka, Japan) in the presence or absence of tissue inhibitor of metalloproteinases-2 (TIMP-2; Dainippon Pharm Co., Toyama, Japan) for 20 h, followed by incubating on DQ-gelatin-coated plates for a period of 4 h. Cells were fixed with 2% paraformaldehyde in PBS. Slides were mounted with coverslips using glycerol/PBS, and examined with at 488 nm (excitation) and 533 nm (emission) using an Olympus LSM-GB200 (Olympus, Tokyo, Japan) equipped with an oil immersion lens. Differential interference contrast (DIC) was used to visualize cells cultured on the matrix.
Western blot analysis
MG-63 (1 × 106) cells were preincubated with 100 ng/ml 5 μM SB203580 (Chemicals Inc., Darmstadt, Germany) for 30 min at 37°C, and MG-63 cells were then placed in serum-free DMEM with 100 μg/ml EMD protein for 48 h. Conditioned media were collected, centrifuged to remove debris, and concentrated in Amicon Centriprep concentrators (Invitrogen) up to 10-fold. Cells were incubated in serum-free Eagle medium with 100 μg/ml EMD protein for 48 h. MG-63 cells prepared as described above were lysed with SDS-sample buffer (80 mM Tris-HCl, 3% SDS, 15% glycerol and 0.01% bromophenol blue) and sonicated briefly in order to shear DNA. Samples were separated on 10% SDS polyacrylamide gels (SDS-PAGE) under reducing conditions. Proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF, Immobilon-P) membranes (Sigma-Aldrich, Inc., St. Louis, MO). Membranes were incubated for 1 h with anti-phospho-p38 antibody (Cell Signaling Technology, Danvers, MA) or anti-p38 antibody (Cell Signaling Technology) in PBS containing 0.05% Tween-20 and 10% Blockace (Dainippon Pharm Co., Toyama, Japan). Peroxidase-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ) was used at a 1:1,000 dilution and immunoreactive bands were visualized using Super Signal west pico chemiluminescent substrate (Pierce Biotechnology Inc., Rockford, IL). Signals on each membrane were analyzed by VersaDoc 5000.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from MG-63 cells cells by RNeasy kit (QIAGEN, Valencia, CA). MG-63 cells were then placed in serum-free DMEM with 100 μg/ml EMD protein for 12 h. After denaturation of total RNA at 70°C for 10 min, cDNA was synthesized with oligo-dT primer by incubating with reverse transcriptase (Qiagen) at 50°C for 30 min. The primers for MMP-2 were 5′-TGGTTTTCCTCCATCCAGTGG-3′ (forward) and 5′-CAGGTTGTCTGAAGTCACTGC-3′ (reverse). The primers for GAPDH were 5′-ACC ACA GTC CAT GCC ATC AC-3′ (forward) and 5′-TCC ACC ACC TTG CTG CTG TA-3′ (reverse). Polymerase chain reactions were performed with Pfu polymerase (Qiagen) initiated by 1 cycle at 95°C for 15 min followed by 30 cycles at 94°C for 45 sec, 55°C for 45 sec, 72°C for 1 min and 1 cycle at 72°C for 10 min for final extension. PCR products were loaded to agarose gel, and stained with ethidium bromide. The bands were analyzed using AlphaImager™ IS-3400 software. Briefly, IDV was measured as the sum of all the pixel values after background correction in each band. The values (AVG) of each band were calculated as IND/AREA, where AREA is the size of the region that was measured. The results are shown the values of AVGMMP1/AVGGAPDH at each time point.
Discussion
In this study, we showed that EMD protein stimulates osteoblasts to degrade gelatin in vitro and that production of MMP-2 is up-regulated in response to EMD protein stimulation. Our results also suggest that adhesion to gelatin enhances production of and activates pro-MMP-2, which is spontaneously produced by osteoblasts. EMD protein stimulates p38 MAPK signaling pathways, which in turn, induce MMP-2 production by osteoblasts. Although previous studies have shown that EMD protein applied to periodontal defects is absorbed into the denuded root dentin surface and induces periodontal tissue regeneration[
14‐
16], the mechanisms of this action remain largely unknown. In this regard, our results show the pivotal role of MMP-2 produced from osteoblasts in response to EMD protein in facilitating periodontal connective regeneration.
MMP-2 plays a crucial role in bone remodeling and mineralization[
17], and several studies have assessed the functional roles of specific MMPs[
18‐
20]. Our results further support the importance of MMPs produced by EMD protein-stimulated osteoblasts in bone remodeling. The observations of residual degradation of ECM in the presence of TIMP-2 may be explained by the involvement of other proteases such as serine protease. Indeed, recent studies have shown that osteoblasts express surface serine protease[
21]. However, the significant inhibition of gelatin degradation by TIMP-2 suggests that EMD protein enhances the production of MMPs on cell surfaces, which facilitates osteoblast-mediated gelatinolysis. In the present study, we demonstrated that MMP-2 is spontaneously produced from unstimulated osteoblasts, and that gelatinase is activated in the presence of EMD protein. In contrast, previous studies have shown that gelatinase MMP-9 was not produced by EMD protein-activated osteoblasts[
10].
EMD protein contains both TGF-β- and BMP-like growth factors, which contribute to the induction of biomineralization during periodontal regeneration[
22]. BMP-2 has been shown to promote bone regeneration in vivo[
23], enhance alkaline phosphatase activity[
24,
25], and increase the production MMPs[
26,
27], thus suggesting that the cytokines present in EMD are necessary to promote bone regeneration. EMD protein also activates alkaline phosphatase activity[
28]. It is possible that the TGF-β and BMP in EMD protein activate osteoblasts. Our data show that EMD protein enhances the production MMP-2 by osteoblasts cells and suggest that bone regeneration depends on MMP-2–activated EMD. These papers support our data[
29,
30]. EMD protein have decreased the levels of MMP-1 and MMP-8 in gingival crevicular fluid after flap surgery in vivo[
6].
EMD-induced VEGF production is regulated by p38 MAPK in human gingival fibroblasts[
31].
We also provided evidence identifying p38 MAPK as the predominant pathway for inducing MMP-2 production in EMD-stimulated osteoblasts. Previous studies have shown that this pathway is important to promote the growth of PDL cells stimulated by EMD protein[
32]. Thus, the MAPK family, including the p38 MAPK, ERK and JNK pathways activated by EMD protein, plays a key role in regulating cellular functions required for periodontal regeneration[
33]. EMD-induced VEGF production is regulated by p38 MAPK in human gingival fibroblasts[
31]. p38 MAPK might regulate not only MMPs production, but also cytokine production in EMD stimulated periodontal ligament. Our study also indicates that inhibition of EMD protein-induced production of MMP-2 leads to significant reductions in the generation of active MMP-2, further supporting the notion that MMP-2 acts as a degradation of gelatin for MMP-2 in EMD protein-stimulated osteoblasts.
In summary, our results suggest a model in which MMP-2 plays a role in periodontal connective tissue remodeling by degrading matrix proteins and/or by activating a potent gelatinase MMP-2 in osteoblasts.
Conclusion
EMD protein induces MMP-2 production via the p-38 MAPK-activating signalling pathways in osteoblasts, thereby stimulating degradation of the surrounding collagen, resulting in changes in ECM structure and promoting periodontal regeneration.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SG participated in planning and designing the study, in the data analysis and drafting of the manuscript. HI performed most of the laboratory work and participated in the data analysis. OS, YU, ED and TI participated in the data analysis. All authors have read and approved the final manuscript.