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RESEARCH REPORT |
1 Department of Periodontics and Endodontics, and
2 Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan;
3 Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical School, 1397-1 Yamane Hidaka, Saitama 350-1241, Japan; and
4 Department of Biologic and Materials Science, School of Dentistry, University of Michigan, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA;
* corresponding author, suzuki-shin{at}tsurumi-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: enamel matrix derivative gel (Emdogain®Gel) • transforming growth factor-ß (TGF-ß) • bone morphogenetic protein (BMP) • luciferase reporter assay
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, it has been suggested that enamel extracts have bioactive properties, bone morphogenetic protein (BMP)-like activity (Iwata et al., 2002) and transforming growth factor-ß (TGF-ß)-like activity (Kawase et al., 2001). Therefore, we hypothesized that the osteoinductive growth factors, BMP and TGF-ß, may be involved in Emd-Gel. In this study, they were examined by the luciferase reporter assay.
The plasminogen activator inhibitor type-1 (PAI-1) has been used as a marker for TGF-ß-induced transcription (Dong et al., 2002), and the induction of PAI-1 gene in Mv1Lu cells has been well-characterized. TGF-ß1 activity has been determined in Mv1Lu cells with the use of the luciferase reporter gene conjugated to the TGF-ß-responsive PAI-1 promoter region (PAI-1 luc) (Saitoh et al., 1996). In contrast, the inhibitor of differentiation-1 (Id1) has been used as a marker for BMP-induced transcription (Katagiri et al., 2002). BMP activity has been determined in C2C12 cells with the use of the luciferase reporter gene conjugated to the BMP-responsive Id1 promoter region (Id1-luc).
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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SDS-Polyacrylamide Gel Electrophoresis
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with the use of a 15% polyacrylamide slab gel containing 1% SDS (Laemmli, 1970). Electrophoresis was carried out with a current of 20 mA for 4 hrs. The gels were stained with 0.125% Coomassie Brilliant Blue R-250 and de-stained with a 7.5% acetic-5% methanol solution. The apparent molecular weights of the protein bands were determined according to Bio-Rad LMW protein standards.
Cell Culture
Mv1Lu mink lung epithelial cells and mouse C2C12 myoblast cells were obtained from the RIKEN Cell Bank (Tsukuba Science City, Ibaraki, Japan). Mv1Lu cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Sigma Chemical Co.), 1% antibiotics [100 U/mL of penicillin-G and 100 µg/mL of streptomycin sulfate (Gibco BRL, Grand Island, NY, USA)] at 37°C in a humidified 5% CO2 atmosphere. C2C12 cells were maintained in DMEM medium containing 15% FBS and 1% antibiotics.
Luciferase Reporter Assays for TGF-ß and BMP Activities
Mv1Lu cells were seeded at 1.5 x 104 cells/well in 96-well plates and incubated for 24 hrs for examination of TGF-ß1 activity. PAI-1-luc conjugated by the TGF-ß-responsive PAI-1 promoter region was transfected in Mv1Lu cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Mv1Lu cells were incubated in serum-free medium for 4 hrs during transient transfection, and the culture medium was replaced with fresh medium containing 2.5% FBS with sample, and then cultured for an additional 24 hrs. The luciferase reporter assay was performed according to the supplier’s protocol (Dual-Luciferase Reporter Assay System, Promega Co., Madison, WI, USA) with a MiniLumat LB 9506 (BERTHOLD GmbH & Co., Bad Wildbad, Germany), for measurement of luciferase activity.
We also examined BMP2 activity using Id1-luc (Katagiri et al., 2002) conjugated by the BMP2-responsive Id1 promoter region in C2C12 cells, which were seeded at 1.0 x 104 cells/well in 96-well plates.
Statistical Analysis
All values are represented as mean ± standard error (SE). Statistical significance was determined by an unpaired Student’s t test, and p < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), as well as TGF-ß1 (Fig. 1A). This indicated that TGF-ß-like activity was contained in Emd-Gel.
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). However, no BMP-like activity was recognized in the unfractionated Emd-Gel (Fig. 1D).
Luciferase Reporter Assays for Fractionated Emd-Gel
We fractionated Emd-Gel using a column of Sephadex G-100 to separate the growth factors (Fig. 2A). The corresponding SDS-PAGE profiles from fractions 1 to 22 are shown in Fig. 2B. This result showed that Emd-Gel mainly contains amelogenins and their derivatives (Fig. 2B).
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). The SDS-PAGE profiles showed 10- to 25-KDa peptides in fractions 4 to 13 (Fig. 2B). The molecular size corresponded to that of TGF-ß and BMP family proteins.
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, 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, it has been reported that EMD contains TGF-ß-like activity (Kawase et al., 2001); therefore, we examined the TGF-ß activity in Emd-Gel using the TGF-ß-specific reporter gene assay. The results showed that TGF-ß-like growth factor was contained in Emd-Gel (Figs. 1B, 3). It has been reported that TGF-ß1 may play an important role in the modulation of tissue formation and development of the periodontium (Gao et al., 1998). Furthermore, TGF-ß1 is mitogenic to periodontal ligament cells (Brady et al., 1998) and inhibits epithelial cell proliferation (Kawase et al., 2002). Based on these results, the TGF-ß-like growth factor in Emd-Gel may have an effect similar to that of TGF-ß1 in periodontal regeneration.
Porcine enamel extracts have biomineralization-promoting activity, such as BMP-like activity (Iwata et al., 2002). However, in this assay system, BMP activity was not detected in unfractionated Emd-Gel (Fig. 1D). Therefore, we fractionated Emd-Gel using a column of Sephadex G-100 equilibrated with 4 M guanidine solution (Fig. 2A) to separate the proteins mixed with propylene glycol alginate (PGA). We then examined each fraction to detect the BMP activity in fractionated Emd-Gel. The results showed that Emd-Gel contained BMP-like growth factor from fractions 4 to 6, and these fractions contained proteins of 20 kDa, similar to BMP (Bessho et al., 1991) (Figs. 2B, 3). It has been reported that the BMP family may play an important role in biomineralization (Wozney et al., 1988; Katagiri et al., 1990; Yamashiro et al., 2003). The BMP-like growth factor in Emd-Gel may stimulate osteogenesis and cementogenesis during periodontal regeneration.
TGF-ß1 potentiated the inhibitory effect of BMP2 (Katagiri et al., 1994; De Jong et al., 2002). Therefore, we investigated the cross-talk of TGF-ß1 and BMP2. These results showed that the signal transduction of BMP2 was inhibited by TGF-ß1 (Fig. 4A). Also, the signal transduction of BMP-like growth factor was inhibited by authentic TGF-ß1 or TGF-ß-like growth factor in Emd-Gel (Fig. 4B). However, the signal transduction of TGF-ß1 was not suppressed by BMP2 (data not shown). It has been reported that the inhibitory Smad6 and 7 exert a negative feedback effect by competing with the receptor-activated Smads (R-Smads: Smads 1, 2, 3, and 5) (Imamura et al., 1997; Nakao et al., 1997). Smad7 inhibits the signaling by both TGF-ß and BMP, whereas Smad6 inhibits TGF-ß signals less effectively (Mochizuki et al., 2004). Therefore, the signal transduction of BMP-2 was inhibited by TGF-ß1, though the signal transduction of TGF-ß1 was not suppressed by BMP-2. Also, Smads 1, 5, and 8 are activated by BMP receptors, whereas Smads 2 and 3 are activated by TGF-ß and activin receptors (Miyazono, 1999). Common mediator Smads (Co-Smad: Smad4) and activin receptor-like kinase (ALK) 1, which is a type I receptor and activates smads 1 and 5, are shared by BMP and TGF-ß signaling pathways (Miyazono, 1999; Derynck and Zhang, 2003). Therefore, it is considered that TGF-ß cannot completely inhibit the activities of BMP.
Another TGF-ß-like activity was detected in the low-molecular region (fractions 19-21) on the G-100 column chromatogram (Figs. 2B, 3). It has been reported that small peptides of amelogenin have bioactivity (Veis et al., 2000). We could not detect such peptides in fractions 19-21. Further investigation will be required to confirm the small peptides.
These results suggest that several growth and differentiation factors exist in Emdogain® Gel (Emd-Gel), which contains TGF-ß- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration.
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ACKNOWLEDGMENTS |
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Received May 6, 2004; Last revision February 27, 2005; Accepted March 22, 2005
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