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