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RGD-CAP (βig-h3) Exerts a Negative Regulatory Function on Mineralization in the Human Periodontal Ligament
S. Ohno1,*,
T. Doi1,
K. Fujimoto2,
C. Ijuin1,
N. Tanaka1,
K. Tanimoto1,
K. Honda1,
M. Nakahara1,
Y. Kato2 and
K. Tanne1
1 Departments of Orthodontics and
2 Biochemistry, Hiroshima University Faculty of Dentistry, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan;
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Figure 1. Expression of RGD-CAP in the PDL. Western blot analysis of RGD-CAP in human PDL in the presence of β-mercaptoethanol (β-ME; 0-5.0%).
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Figure 2. RGD-CAP mRNA level and ALP activity cultured PDL cells. After the PDL cells became confluent, the cells maintained in Medium A were treated with 10–8 M dexamethasone (Dex) or 10–8 M 1 ,25-dihydroxyvitamin D3 (vitamin D3) for 0-11 days. The rate of increase in RGD-CAP mRNA expression determined by real-time PCR is shown as a bar. The dotted line indicates the level of RGD-CAP mRNA on day 0. Levels of ALP activity in these cultures were evaluated by measurement of the absorbance at 405 nm and are shown as lines. Values are averages ± SD of triplicate cultures.
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Figure 3. Effects of RGD-CAP on mineralization of PDL cells. (A) The PDL cells were seeded confluently on the 35-mm dishes coated with recombinant RGD-CAP (20 µg/mL), maintained in Medium A for 1-5 days. ALP activity was measured following the method described in MATERIALS & METHODS. Values are averages ± SD of triplicate cultures. (B,C) A 20-µg/mL quantity of recombinant RGD-CAP in the solution buffer (PBS containing 4 M urea) or solution buffer (control) was added to the mineralizing medium (MM) of PDL cell cultures every 2 days. Total RNA was extracted on day 11, and RT-PCR was performed. Ethidium bromide staining pattern of PCR products of type I collagen (Col I), bone sialoprotein (BSP), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was shown in panel (B). We determined the relative mRNA expression of Col I or BSP by dividing the densitometric value of RT-PCR products of each transcript by that of G3PDH. Alizarin red staining was performed for the cells cultured on day 21, and the number of bone nodules was counted (C). **p < 0.01.
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Journal of Dental Research, Vol. 81, No. 12,
822-825 (2002)
DOI: 10.1177/154405910208101205
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