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doi: 10.1242/10.1242/dev.00110

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Regulation of the chondrocyte phenotype by ß-catenin

¹ Department of Life Science, Kwangju Institute of Science and Technology, Gwangju 500-712, Korea
² Department of Orthopaedic Surgery, Wonkwang University School of Medicine, Iksan 570-711, Korea
³ Laboratory of Developmental Biology and Genomics, College of Veterinary Medicine, Seoul National University, Seoul, Korea
⁴ TG Biotech, Kyungpook National University, Daegu 702-701, Korea

View larger version (71K): [in a new window]   Fig. 1. Expression pattern of ß-catenin in developing wing buds of chicken embryo. (A) Type II collagen and ß-catenin in wing buds of day 5 and 6 chicken embryos were detected by immunohistochemistry. Accumulation of sulfated proteoglycan was detected by Alcian Blue staining. (B) Higher magnification in area between differentiating cartilage (C) and non-cartilage (NC) at day 6 embryo.

View larger version (51K): [in a new window]   Fig. 2. Expression of ß-catenin negatively regulates chondrogenesis in vitro. (A) Mesenchymal cells from chicken embryo were maintained as a micromass culture for 4 days, and distribution of type II collagen and ß-catenin was detected by immunocytochemical staining of cross-sections of a micromass culture spot (top and middle). Subcellular localization of ß-catenin was determined in day-2 culture (bottom). (B) Mesenchymal cells were maintained as micromass culture for the indicated period to induce chondrogenesis (top). Alternatively, the cells were cultured for 4 days in the presence of vehicle alone as a control, 10 nM phorbol ester (PMA), 10 ng/ml epidermal growth factor (EGF), 10 µM SB203580 (SB) or 10 µM PD98059 (PD) (middle and bottom). Type II collagen, ß-catenin and N-cadherin were detected by Western blotting (middle). Synthesis of sulfated proteoglycan was determined by Alcian Blue staining and quantified (bottom). (C) Chondrifying mesenchymal cells during micromass culture were treated with 5 mM NaCl as a control or 5 mM LiCl for indicated periods (top), for 5 days (middle) or for 5 days with the indicated concentrations of LiCl (bottom). Expression of type II collagen, ß-catenin, N-cadherin and phosphorylated GSK-3ß was determined by western blotting (top and middle panels). Accumulation of sulfated proteoglycan at day 4 culture was determined by Alcian Blue staining and quantified (bottom). ERK-2 was detected as loading control of blots.

View larger version (30K): [in a new window]   Fig. 3. Expression patterns of ß-catenin during de- and redifferentiation of chondrocytes. (A) Articular chondrocytes were maintained as a serial monolayer up until the indicated passages (top) or P0 chondrocytes were treated with 1 µM retinoic acid (RA) (middle) or 5 ng/ml IL1ß (bottom) for 72 hours. Expression patterns of type II collagen and ß-catenin were determined by western blot analysis. (B) Chondrocytes were untreated or treated with IL1ß or RA or P0 chondrocytes were passed to P4 (P4). Accumulation of sulfated proteglycan was determined by Alcian Blue staining (top) and type II collagen transcript level was determined by northern blot (NB) analysis (bottom). (C) Chondrocytes were cultured as monolayer (M) to P0 or to P4. P4 cells were detached and three-dimensionally cultured for 4 days in alginate gel (Alginate). Type II collagen and ß-catenin expression was determined by western blot analysis. ERK-2 was detected as loading control of blots.

View larger version (29K): [in a new window]   Fig. 4. Post-translational accumulation of ß-catenin during de-differentiation of chondrocytes. (A) Chondrocytes were untreated or treated with 5 ng/ml IL1ß or 1 µM RA for 72 hours, and ß-catenin and phosphorylated GSK-3ß was determined by western (WB) or northern (NB) blotting. (B) Chondrocytes were treated with the indicated concentrations of LiCl for 48 hours. ß-Catenin, type II collagen and phosphorylated GSK-3ß was determined by western blotting. (C) Chondrocytes were treated with the indicated concentrations of MG132 for 48 hours. ß-Catenin and type II collagen was determined by western blotting. ERK-2 was detected as loading control of blots.

View larger version (42K): [in a new window]   Fig. 5. ß-Catenin function as a cytoskeletal component is not necessary for phenotype modulation of chondrocytes. (A) Rabbit articular chondrocytes were treated with the indicated concentrations of RA or IL1ß for 72 hours. Expression of N-cadherin and -catenin was determined by western blotting (top). Chondrocytes were untreated (Con) or treated with 1 µM RA or 5 ng/ml IL1ß for 72 hours. N-cadherin was detected by western blotting from the samples precipitated with ß-catenin immuncomplex. Alternatively, N-cadherin was immunoprecipitated and ß-catenin was detected by western blot analysis (bottom). (B,C) Chondrocytes were treated with vehicle alone as a control, RA or IL1ß. ß-Catenin in Triton X-100 insoluble fraction was detected by western blotting (B) or indirect immunofluorescence microscopy (C). (D) Chondrocytes were untreated or treated with RA or IL1ß. The cells were fixed and immunostained for F-actin with palloidin-rhodamine and analyzed by immunofluorescence microscopy.

View larger version (73K): [in a new window]   Fig. 6. ß-Catenin function as a nuclear signaling molecule is sufficient to cause phenotype loss of chondrocytes. (A) Chondrocytes were treated with vehicle alone as a control, 1 µM RA or 5 ng/ml IL1ß for 72 hours. The distribution of ß-catenin was determined by immunofluorescence microscopy. (B) Chondrocytes were transfected with active (TOPFlash) or inactive (FOPFlash) TCF/LEF reporter gene for ß-catenin and treated with vehicle alone as a control (Con), RA, or IL1ß, and TCF/LEF reporter activity was monitored (upper panel). ß-Catenin protein in nuclear preparation or Jun and connexin 43 (Cx43) in whole cell lysates were detected by western blotting from chondrocytes treated with RA or IL1ß.

View larger version (37K): [in a new window]   Fig. 7. Ectopic expression of S37A ß-catenin causes phenotype loss of chondrocytes. (A) Chondrocytes were transfected with empty vector (Con) or S37A ß-catenin. After 72 hours incubation, expression of type II collagen, ß-catenin, Jun and connexin 43 (Cx43) was determined (left). TCF/LEF activity was determined by TOPFlash assay and accumulation of sulfated proteoglycan was determined by Alcian Blue staining and quantified in control cells (C) or cells transfected with S37A ß-catenin (S37A) (right). (B) Type II collagen and ß-catenin were double stained in chondrocytes transfected with S37A ß-catenin and analyzed by immunofluorescence microscopy.

View larger version (56K): [in a new window]   Fig. 8. Expression pattern of Jun during chondrogenesis in vivo and in vitro. (A) Type II collagen and Jun in wing buds of day 5 and 6 chicken embryos were detected by immunohistochemistry. (B) Mesenchymal cells from chicken embryo were maintained as a micromass culture for 4 days, and distribution of type II collagen and Jun was detected by immunocytochemical staining of cross-sections of a micromass culture spot. (C) Mesenchymal cells were maintained as micromass culture for the indicated period to induce chondrogenesis. (D) Alternatively, the cells were cultured for 4 days in the presence of vehicle alone as a control (Con), 10 nM phorbol ester (PMA), 1 µM Go6976 (Go) or 10 µM PD98059 (PD). Type II collagen, ß-catenin and Jun were detected by western blotting.