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doi: 10.1242/10.1242/jcs.00098

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A cell line with characteristics of the periodontal ligament fibroblasts is negatively regulated for mineralization and Runx2/Cbfa1/Osf2 activity, part of which can be overcome by bone morphogenetic protein-2

Yoshinori Saito1,2,*, Tatsuya Yoshizawa1,*, Fumio Takizawa1,2, Mika Ikegame1,3, Osamu Ishibashi1, Kazuhiro Okuda2, Kohji Hara2, Kotaro Ishibashi4, Masuo Obinata5 and Hiroyuki Kawashima1,{ddagger}

1 Divisions of Cell Biology and Molecular Pharmacology, Niigata University Graduate School of Medical and Dental Sciences, 5274 2-Bancho, Gakkocho-dori, Niigata-city, Niigata 951-8514, Japan
2 Division of Periodontology, Niigata University Graduate School of Medical and Dental Sciences, 5274 2-Bancho, Gakkocho-dori, Niigata-city, Niigata 951-8514, Japan
3 Divisions of Anatomy and Cell Biology of the Hard Tissue, Niigata University Graduate School of Medical and Dental Sciences, 5274 2-Bancho, Gakkocho-dori, Niigata-city, Niigata 951-8514, Japan
4 Daiichi Pharmaceutical Co. Ltd., 1-16-13 Kita-kasai, Edogawa-ku, Tokyo 134-8630, Japan
5 Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai, Miyagi 980-8575, Japan



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  		Fig. 1. Fibroblastic PDL cells express ALPase activity and genes for type I collagen, periostin and Runx2/Osf2, but not genes for osteocalcin and BSP. (A) ALPase staining of mouse mandibula. (B) Higher magnification of the inset in A. (C-G) In situ hybridization studies. The antisense probes used were C, type I collagen; D, periostin, E, Runx2/Cbfa1/Osf2; F, osteocalcin (OCN); and G, BSP. (H) A schematic drawing of the tissue section used. B-G show a tissue section roughly corresponding to the schematic drawing shown in H. Signals in C-G disappeared when the corresponding sense probes were used (data not shown). Note that expression of the periostin gene, originally cloned as an osteoblast-specific marker, in the fibroblastic PDL cells is much higher than in the osteoblasts lining the surface of alveolar bone (D). The expression level of Runx2/Cbfa1/Osf2 gene was comparable in PDL, osteoblasts and odontoblasts, but was zero in the cementoblasts (E). The expression of the OCN gene is highest in the odontoblasts, followed by osteoblasts and then by cementoblasts. Expression of PDL cells is undetectable (F). BSP expression is detected in the cementoblasts and osteoblasts but not in PDL cells (G). Original magnification: A, x8; B-G, x40. See text for details.

 


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  		Fig. 2. RT-PCR analysis demonstrates that the PDL-L2 has gene expression identical to that of PDL cells in vivo. (A) MC3T3-E1 (U) represents MC3T3-E1 cells at 80% confluence; MC3T3-E1 (D) represents MC3T3-E1 cells at a fully differentiated stage (mineralization stage); PDL-L2 and NIH3T3 represent cells at confluence. Note that the expression level of periostin decreases with maturation of osteoblastic MC3T3-E1 cells, and it is higher in PDL-L2. The data are consistent with Fig. 1D. Runx2/Cbfa1/Osf2 is expressed in both PDL-L2 and MC3T3-E1 cells, whereas OCN and BSP are absent in PDL-L2 but present in MC3T3-E1. These are also consistent with Fig. 1E-G. (B) Nuclear extracts (N.E.) of PDL-L2 and MC3T3-E1 or cell lysates (C.L.) of C3H10T1/2 were electrophoresed on 8% SDS-PAGE and then subjected to immunoblot analysis with an antibody against Runx/Cbfa1/Osf2. Runx2/Osf2 is detected in the PDL-L2 cells, although its expression level is one-sixth of that in MC3T3-E1 cells. Since C3H10T1/2 cells do not express Runx2/Osf2, cells transfected with pcDNA3-Runx2/Osf2 were used for the control. Note that 20-fold dilution of cell lysate gave a comparable expression level of protein to the endogenous Runx2/Osf2 in the nuclear extract of PDL-L2. See text for details.

 


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  		Fig. 3. rhBMP-2 induces ALPase activity in PDL-L2 cells in vitro. Cells were cultured either in the normal medium or the differentiation medium. After 3 days of incubation, cells were given 250 ng/ml rhBMP-2 or vehicle and incubated for an additional 3 days. Cells were then fixed and subjected to ALPase staining. Consistent with previous reports, MC3T3-E1 expressed more ALPase activity as the incubation increased, and this was markedly enhanced by rhBMP-2. ALPase staining of PDL-L2 is faint and scarce in the absence of rhBMP-2. However, addition of rhBMP-2 is markedly induced to a level close to that of unstimulated MC3T3-E1 cells. Note that fibroblastic NIH3T3 cells did not respond to rhBMP at all. Original magnification was x4. See text for details.

 


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  		Fig. 4. PDL-L2 cells produce mineralized nodules in response to rhBMP-2. Cells were cultured for 3 days in the differentiation medium. Cells were then given either 250 ng/ml rhBMP-2 or vehicle and incubated for an additional 3 days. Cells were cultured for a further 25 days without any treatment in the differentiation medium. The medium was changed every 3 days. Cultured cells were fixed and subjected to Arizalin red-S staining (A). Original magnification was x4. After being photographed, each culture dish was subjected to the destaining process (see text for detail), and the Arizalin red-S concentration was determined by absorbance at 562 nm. Note that the extent of mineralization of PDL cells, a mixture of various cells from PDL, is even higher than that of MC3T3-E1 cells (B). See text for details.

 


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  		Fig. 5. Transcriptional activity of Runx2/Cbfa1 is suppressed in the PDL-L2 cells. (A) PDL-L2 cells were cultured in the differentiation medium (D) with or without 250 ng/ml rhBMP-2 and gene expression was examined by RT-PCR. (B) Each cell line was transfected with p6OSE-2-Luc, and luciferase activity was determined. (C) Each cell line was transfected with p6OSE-2-Luc together with pcDNA3-Runx2/Osf2 or pcDNA3, and luciferase activity was determined. Each assay was carried out in a duplicate or triplicate. Each set of data is a representative of at least two independent experiments. See text for details.

 




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