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Amelogenin-mediated Regulation of Osteoclastogenesis, and Periodontal Cell Proliferation and Migration

¹ Functional Genomics Section and
² Cell Biology Section, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive, MSC 4395, Bldg. 30, Room 122, Bethesda, MD 20892, USA;
³ Cartilage Biology and Orthopedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA; and
⁴ Department of Anatomy and Cell Biology, University of Pennsylvania School of Dental Medicine, Philadelphia, PA, USA

View larger version (57K): [in a new window]   Figure 1. LRAP inhibits osteoclastogenesis. (A) Number of osteoclasts (tartrate-resistant alkaline phosphatase [TRAP])-positive multinucleated cells, as indicated by arrows in the subsequent panels in the co-cultures of wild-type (WT) osteoclast progenitor (bone marrow cells) and amelogenin-KO (KO) or WT-cementoblast/periodontal ligament cells treated with 10–8 M of 1,25(OH)2D3 for 7 days. Increased numbers of osteoclasts were observed in the co-cultures of osteoclast progenitor and KO cementoblast/periodontal ligament cells (compare panels B and G). (B–K) Osteoclast formation in the co-cultures of WT osteoclast progenitor and KO or WT-cementoblast/periodontal ligament cells treated with LRAP or P172 for 7 days in 96-well plates in the presence of 10–8 M of 1,25(OH)2D3. Different amounts of LRAP and P172 (1, 10, and 100 ng/mL) were added in the culture medium. TRAP staining of co-cultures of cementoblast/periodontal ligament cells from KO mice (B–E) and WT mice (G–J) treated with either LRAP or P172 as indicated. Data are expressed as the mean ± SD (n = 18). **P < 0.01. Scale bar: 100 µm.

View larger version (37K): [in a new window]   Figure 2. LRAP inhibits the osteoclastogenic pathway. (A,B) RANKL and osteoprotegerin (OPG) mRNA levels as measured by RT-PCR analysis of total RNA extracted from cementoblast/periodontal ligament cells treated either with 100 ng/mL of LRAP (A) or P172 (B) for 0, 12, 24, 48, or 72 hrs. GAPDH was used as the internal control. WT, wild-type mice; KO, amelogenin-KO mice.

View larger version (39K): [in a new window]   Figure 3. Both LRAP and P172 induce the proliferation of cementoblast/periodontal ligament cells. (A) Amelogenin-KO (KO) cementoblast/periodontal ligament cells showed reduced proliferation. **P < 0.01. (B,C) Increased proliferation rate of KO cementoblast/periodontal ligament cells treated with either LRAP or P172. Amelogenin-KO cementoblast/periodontal ligament cells were cultured with various concentrations of LRAP (B) or P172 (C) for 1, 3, and 5 days. *P < 0.05 vs. day 0, **P < 0.01 vs. day 0, #P < 0.05 vs. 0 ng/mL group, and ##P < 0.01 vs. 0 ng/mL group. (D,E) Wild-type (WT) cementoblast/periodontal ligament cells were cultured with various concentrations of LRAP (D) or P172 (E) for 1, 3, or 5 days. Data are expressed as the mean ± SD (n = 3). *P < 0.05 vs. day 0, **P < 0.01 vs. day 0, #P < 0.05 vs. 0 ng/mL group, and ## P < 0.01 vs. 0 ng/mL group.

View larger version (32K): [in a new window]   Figure 4. Both LRAP and P172 promote migration of cementoblast/periodontal ligament cells. (A) Amelogenin-KO (KO) cementoblast/periodontal ligament cells exhibited a reduced cell migration rate. (B) Addition of the conditioned medium from the three-day cultures of wild-type cementoblast/periodontal ligament cells increased the migration rate of amelogenin KO-cementoblast/periodontal ligament cells. (C,D) Increased cell migration rates in the WT and amelogenin KO-cementoblast/periodontal ligament cells cultured with 100 ng/mL LRAP (C) and P172 (D) for 12 hrs. Data are expressed as the mean ± SD (n = 3) of the µm distance covered by cementoblast/periodontal ligament cells. *P < 0.05 vs. day 0 and **P < 0.01 vs. day 0.