Inhibition of beta-catenin signaling in articular chondrocytes results in articular cartilage destruction

Abstract

Objective: Osteoarthritis is a degenerative joint disease whose molecular mechanism is currently unknown. Wnt/beta-catenin signaling has been demonstrated to play a critical role in the development and function of articular chondrocytes. To determine the role of beta-catenin signaling in articular chondrocyte function, we generated Col2a1-ICAT-transgenic mice to inhibit beta-catenin signaling in chondrocytes.

Methods: The expression of the ICAT transgene was determined by immunostaining and Western blot analysis. Histologic analyses were performed to determine changes in articular cartilage structure and morphology. Cell apoptosis was determined by TUNEL staining and the immunostaining of cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) proteins. Expression of Bcl-2, Bcl-x(L), and Bax proteins and caspase 9 and caspase 3/7 activities were examined in primary sternal chondrocytes isolated from 3-day-old neonatal Col2a1-ICAT-transgenic mice and their wild-type littermates and in primary chicken and porcine articular chondrocytes.

Results: Expression of the ICAT transgene was detected in articular chondrocytes of the transgenic mice. Associated with this, age-dependent articular cartilage destruction was observed in Col2a1-ICAT-transgenic mice. A significant increase in cell apoptosis in articular chondrocytes was identified by TUNEL staining and the immunostaining of cleaved caspase 3 and PARP proteins in these transgenic mice. Consistent with this, Bcl-2 and Bcl-x(L) expression were decreased and caspase 9 and caspase 3/7 activity were increased, suggesting that increased cell apoptosis may contribute significantly to the articular cartilage destruction observed in Col2a1-ICAT-transgenic mice.

Conclusion: Inhibition of beta-catenin signaling in articular chondrocytes causes increased cell apoptosis and articular cartilage destruction in Col2a1-ICAT- transgenic mice.

Figure 1. Generation of Col2a1-ICAT–transgenic mice and verification of signaling phenotype

Two lines of Col2a1-ICAT–transgenic mice were established. The expression of the FLAG-ICAT transgene and the effect of inhibitor of β-catenin and T cell factor (ICAT) on β-catenin signaling in chondrocytes were examined. a and b, Immunofluorescence staining was performed using tissue sections from long bones of 2-week-old mice (a) and 6-month-old mice (b). Safranin O–fast green–stained histologic sections from 2-week-old mice and Alcian blue/hematoxylin and eosin (H&E)–stained histologic sections from 6-month-old mice were used as controls. Using the anti-FLAG M2 antibody, the expression of the FLAG-ICAT transgene was detected in articular chondrocytes of Col2a1-ICAT–transgenic mice but not in those of their wild-type (WT) littermates. c, To determine changes in β-catenin signaling in Col2a1-ICAT–transgenic mice, primary sternal chondrocytes isolated from 3-day-old Col2a1-ICAT–transgenic mice and their WT littermates were transfected with Top-flash reporter construct and treated for 48 hours with glycogen synthase kinase 3β inhibitor, BIO (1 µM). Values are the mean and SEM. BIO stimulated Top-flash reporter activity in WT chondrocytes (* = P < 0.05 versus unstimulated WT chondrocytes, by unpaired t-test). In transgenic mouse chondrocytes, the basal reporter activity was reduced and the stimulatory effect of BIO was completely inhibited. d and e, To further demonstrate FLAG-ICAT transgene expression in vitro, we performed Western blot (d) and immunostaining (e) assays using primary sternal chondrocytes derived from 3-day-old Col2a1-ICAT–transgenic mice and their WT littermates. Western blot and immunostaining data showed that FLAG-ICAT was detected only in chondrocytes derived from Col2a1-ICAT–transgenic mice and not in those from their WT littermates. f, The expression of β-catenin signaling downstream genes, Tcf3, Tcf4, and Lef, was examined by reverse transcriptase–polymerase chain reaction assays. The mRNA expression of Tcf3, Tcf4, and Lef was reduced in chondrocytes derived from Col2a1-ICAT–transgenic mice. (Original magnification × 10 in a; × 20 in b and e.) DAPI = 4′, 6-diamidino-2-phenylindole.

Figure 2. Articular cartilage destruction in 6-month-old Col2a1-ICAT–transgenic mice

a, Changes in knee joint structure of Col2a1-ICAT–transgenic mice were analyzed by histology using Alcian blue/H&E and orange G staining. Mild cartilage degeneration was observed at the articular surface of knee joints in 7 of 10 6-month-old Col2a1-ICAT–transgenic mice. In the weight-bearing area, the articular layer was thinner or even completely missing in transgenic mice. Chondrocyte clusters (arrow) were found in Col2a1-ICAT–transgenic mice. No articular cartilage damage was found in 6-month-old WT mice. Boxed areas in upper panels are shown at higher magnification in lower panels. b–d, Histologic and histomorphometric analyses showed that articular area (b and c) and articular chondrocyte numbers (d) were significantly reduced in Col2a1-ICAT–transgenic mice. Values are the mean and SEM. * = P < 0.05 versus WT mice, by unpaired t-test (n = 6 mice per group). (Original magnification × 10.) ROI = region of interest (see Figure 1 for other definitions).

Figure 3. Progressive destruction of articular cartilage in 9- and 12-month-old Col2a1-ICAT–transgenic mice

a, Histologic analysis showed moderate destruction of articular cartilage tissue, and defects were extended to the calcified cartilage area in 6 of 9 9-month-old Col2a1-ICAT–transgenic mice (arrow). b, Severe destruction of articular cartilage and subchondral bone was detected in 6 of 11 12-month-old Col2a1-ICAT–transgenic mice. In contrast, only minor articular cartilage damage was found in <10% of 9- and 12-month-old wild-type mice (10 mice were analyzed). (Original magnification × 10.)

Figure 4. Ectopic and new bone formation in articular cartilage area of Col2a1-ICAT–transgenic mice

a, Radiography shows ectopic bone formation (arrow) in the knee joint of a 15-month-old Col2a1-ICAT–transgenic mouse. b, Histologic analysis reveals the complete loss of articular tissue at the weight-bearing area in a 15-month-old transgenic mouse. The destruction extends to the subchondral bone region. Boxed areas in upper panels are shown at higher magnification in lower panels. The high-magnification picture shows the deep cleft (black arrows) and new woven bone formation (blue arrow) in the knee joint area of a 15-month-old Col2a1-ICAT–transgenic mouse. (Original magnification × 10.)

Figure 5. Increased cell apoptosis in articular cartilage of Col2a1-ICAT–transgenic mice

a, Cell apoptosis was determined by TUNEL staining. A significant increase in numbers of apoptotic cells was found in articular chondrocytes of 6-month-old Col2a1-ICAT–transgenic mice (arrows). DAPI staining was used as a positive control. The Alcian blue/H&E–stained histologic pictures from the same tissue blot are also shown. b, Quantification of apoptotic cells showed a 75% increase in the number of apoptotic cells in Col2a1-ICAT–transgenic mice. Values are the mean and SEM. * = P < 0.05 versus WT mice, by unpaired t-test (n = 3 mice per group). c and d, The immunostaining of cleaved caspase 3 (c) and poly(ADP-ribose) polymerase (PARP) (d) proteins was performed using knee joint sections derived from 6-month-old WT and Col2a1-ICAT–transgenic mice. The immunostaining of both cleaved caspase 3 and PARP was significantly increased in articular cartilage of Col2a1-ICAT–transgenic mice (arrows). (Original magnification × 20.) ROI = region of interest (see Figure 1 for other definitions).

Figure 6. Changes in Bcl-2, Bcl-x_L, and Bax expression and caspase 9 and caspase 3/7 activities in primary sternal chondrocytes of Col2a1-ICAT–transgenic mice and in primary chicken and porcine articular chondrocytes

a, Primary sternal chondrocytes were isolated from 3-day-old Col2a1-ICAT–transgenic mice and their WT littermates. The expression of Bcl-2, Bcl-x_L, and Bax proteins was detected by Western blot analysis. The expression levels of Bcl-2 and Bcl-x_L proteins were decreased in primary sternal chondrocytes derived from Col2a1-ICAT–transgenic mice, while the expression level of Bax protein was increased. b, Caspase 9 and caspase 3/7 activities were measured and found to be significantly increased in primary sternal chondrocytes derived from Col2a1-ICAT–transgenic mice. c and d, Caspase 9 and caspase 3/7 activities were also measured in primary chicken (c) and porcine (d) articular chondrocytes. Wnt-3A (100 ng/ml) significantly inhibited caspase 9 activity and Dkk-1 (1 µg/ml) stimulated caspase 3/7 activity in chicken articular chondrocytes after 48-hour treatment. In contrast, Wnt-3A (100 ng/ml, 48-hour treatment) inhibited both caspase 9 and caspase 3/7 activities in porcine articular chondrocytes. Values in b–d are the mean and SEM. * = P < 0.05 versus WT mice or versus control (Cont), by unpaired t-test (n = 4 wells per group). e, Bax protein expression was reduced by 48-hour treatment with Wnt-3A (100 ng/ml) in primary porcine articular chondrocytes. See Figure 1 for other definitions.