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First published online December 31, 2003
doi: 10.1242/10.1242/jcs.00866

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Runx2 deficiency in chondrocytes causes adipogenic changes in vitro

Hirayuki Enomoto^1,*, Tatsuya Furuichi^1,*, Akira Zanma², Kei Yamana², Carolina Yoshida^1,3, Satoru Sumitani¹, Hiroyasu Yamamoto¹, Motomi Enomoto-Iwamoto⁴, Masahiro Iwamoto⁴ and Toshihisa Komori^1,

¹ Department of Molecular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
² Teijin Institute for Biomedical Research, Teijin Ltd., Hino, Tokyo 191-8512, Japan
³ Department of Orthodontics and Dentofacial Orthopedics, Osaka University Faculty of Dentistry, Suita, Osaka 565-0871, Japan
⁴ Department of Orthopaedic Surgery, Thomas Jefferson Medical College, Philadelphia PA 19107, USA

View larger version (79K): [in a new window]   Fig. 1. Morphological changes of chondrocytes in culture. Chondrocytes isolated from the ribs of wild-type (A-C) and Runx2–/– (D-F) embryos at E18.5 were inoculated in DMEM/F12 hybrid medium containing 10% FBS at a density of 1x105/plate in 24-well plates, and cultured for up to 9 days. Phase-contrast images at days 3, 6 and 9 (with day 0 as the day of plating) are shown. Magnification, x50.

View larger version (140K): [in a new window]   Fig. 2. Adipocyte differentiation of Runx2–/– chondrocytes during culture. Chondrocytes isolated from the ribs of wild-type (D, F) and Runx2–/– (A-C,E,F) embryos at E18.5 were inoculated at a density of 1x105/plate in 24-well plates, and cultured for up to 9 days. (A) Immunocytochemistry using anti-type II collagen antibody of Runx2–/– chondrocytes 1 day after plating. (B,C) Runx2–/– chondrocytes were stained with Alcian blue (B) or with Alcian blue and oil red O (C) at 6 days after plating. Cells with vacuoles are surrounded by proteoglycan, as shown by staining with Alcian blue (B, arrows). (D-F) Wild-type (D, F) and Runx2–/– (E,F) chondrocytes were stained with oil red O 9 days after plating. Low-magnification views are shown in (F). The plates were also stained with hematoxylin and eosin (H-E) to confirm the presence of the cells (F). Magnification A-E, x50.

View larger version (61K): [in a new window]   Fig. 3. Northern blot analysis for chondrocyte and adipocyte differentiation markers during cell culture of Runx2–/– and wild-type chondrocytes. Expression of chondrocyte and adipocyte differentiation markers in Runx2–/– chondrocytes on the indicated days (with day 0 as the day of plating) (A), and in Runx2–/– (–/–) and wild-type (+/+) chondrocytes on day 12 (B). Chondrocytes isolated from the ribs of Runx2–/– and wild-type embryos at E18.5 were plated at a density of 1x106/dish in 6 cm dishes and cultured in DMEM/F12 hybrid medium containing 10% FBS. Twenty µg of total RNA were loaded in each lane and hybridized with mouse cDNA of type II collagen, type X collagen, PPAR, aP2, and Glut4, and rat cDNA of Pref-1. Gels stained with ethidium bromide are shown in the lowest panels as an internal control.

View larger version (53K): [in a new window]   Fig. 4. Inhibition of adipogenesis by the adenoviral introduction of Runx2 in Runx2–/– chondrocytes. Runx2–/– chondrocytes infected with either EGFP-expressing (GFP) or Runx2-and-EGFP-expressing (Runx2) virus were cultured for 12 days and stained with oil red O (A), cultured for 6 days and harvested for northern blot analysis (B), or cultured for 15 days and harvested for real-time RT-PCR analysis at indicated days (C). Phase-contrast images are shown in (A) (x50). In northern blot analysis, 20 µg of total RNA were loaded in each lane and hybridized with mouse type II collagen or PPAR cDNA (B). The gel stained with ethidium bromide is shown as an internal control. In real-time RT-PCR analysis, Runx2 and type X collagen expression were examined (C). EGFP-expressing and Runx2-and-EGFP-expressing adenovirus infection are shown by open circles and closed circles, respectively. The value of Runx2 or type X collagen in Runx2-and-EGFP-expressing adenovirus infection on 15-day culture was defined as 1, and relative values are shown. Data represent the mean of three wells. *P<0.05 as determined by one-way ANOVA. Similar results were obtained in three independent experiments and representative data are shown.

View larger version (80K): [in a new window]   Fig. 5. Inhibition of adipogenesis of Runx2–/– chondrocytes by soluble differentiation factors. Runx2–/– primary chondrocytes were plated at a density of 1x105 cells/well (5x104 cells/cm2) in 24 multi-well plates. Two days after inoculation, the cells were further cultured for 10 days without (control) or with a factor including TGF-ß (1 ng/ml), retinoic acid (RA) (10–8 M), IL-1ß (10 ng/ml), PDGF (200 ng/ml), bFGF (10 ng/ml), IL-11 (100 ng/ml), PTH (10–7 M), BMP-2 (50 ng/ml), IGF-1 (100 ng/ml) or T3 (10–7 M), and stained with oil red O or Alcian blue. After culture, the cells were replated for the examination of type II collagen expression by immunocytochemistry. Magnification: phase contrast, oil red and Alcian blue, x50; type II collagen, x100.

View larger version (15K): [in a new window]   Fig. 6. Real-time RT-PCR for IL-11 expression in the skeletons of wild-type and Runx2–/– embryos and upregulation of IL-11 expression in Runx2–/– chondrocytes by adenoviral infection of Runx2. (A) IL-11 expression in the skeletons of wild-type embryos (+/+) at E12.5, E13.5, E16.5, and E18.5, and Runx2–/– embryos (–/–) at E18.5. IL-11 expression was examined by real-time RT-PCR. The value in the E12.5 wild-type embryos was defined as 1 and relative values are shown. Data represent the mean of three to five embryos in each group. The IL-11 expression in Runx2–/– skeletons at E18.5 is significantly lower than those in wild-type skeletons at all embryonic stages. P<0.05 as determined by one-way ANOVA. (B) Runx2 and IL-11 expression in Runx2–/– chondrocytes infected with either EGFP-expressing (open columns) or Runx2-and-EGFP-expressing (closed columns) adenovirus. Infected cells were harvested at the indicated times after the onset of viral infection, and Runx2 and IL-11 expression were examined by real-time RT-PCR. The value of Runx2 or IL-11 in Runx2-and-EGFP-expressing adenovirus infection at 48 hours was defined as 1, and relative values are shown. Data represent the mean of three wells. *P<0.05 and **P<0.01 as determined by one-way ANOVA. Similar results were obtained in three independent experiments, and representative data are shown. (C) Synergistic induction of IL-11 mRNA by Runx2 and TGF-ß. Runx2–/– chondrocytes were infected with EGFP-expressing (GFP) or Runx2-and-EGFP-expressing (Runx2) adenovirus. Runx2–/– chondrocytes and Runx2 overexpressing Runx2–/– chondrocytes were treated with TGF-ß (1 ng/ml) for the indicated times. The value of IL-11 in Runx2-and-EGFP-expressing adenovirus infection at 8 hours was defined as 1, and relative values are shown. The induction of IL-11 mRNA by Runx2 was much weaker than that by TGF-ß but Runx2 hugely induced IL-11 mRNA in the presence of TGF-ß. Data represent mean of four wells. *P<0.01 and **P<0.001 as determined by one-way ANOVA. Similar results were obtained in three independent experiments and representative data are shown.