Activation of beta-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult beta-catenin conditional activation mice

Abstract

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/beta-catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for beta-catenin in this disease has not been reported. Because tissue-specific activation of the beta-catenin gene (targeted by Col2a1-Cre) is embryonic lethal, we specifically activated the beta-catenin gene in articular chondrocytes in adult mice by generating beta-catenin conditional activation (cAct) mice through breeding of beta-catenin(fx(Ex3)/fx(Ex3)) mice with Col2a1-CreER(T2) transgenic mice. Deletion of exon 3 of the beta-catenin gene results in the production of a stabilized fusion beta-catenin protein that is resistant to phosphorylation by GSK-3beta. In this study, tamoxifen was administered to the 3- and 6-mo-old Col2a1-CreER(T2);beta-catenin(fx(Ex3)/wt) mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of beta-catenin protein was detected by immunostaining in articular cartilage tissues of beta-catenin cAct mice. In 5-mo-old beta-catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8-mo-old beta-catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in beta-catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp-9, Mmp-13, Alp, Oc, and colX, was significantly increased (3- to 6-fold) in articular chondrocytes derived from beta-catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6-fold increase) in these cells. In addition, we also observed overexpression of beta-catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of beta-catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA-like phenotype. This study provides direct and definitive evidence about the role of beta-catenin in the development of OA.

FIG. 1

TM-induced Cre-recombination in adult articular chondrocytes. Three- and 6-mo-old Col2a1-CreER^T2;R26R mice and Cre-negative control littermates were administered with TM (1 mg/10 g body weight, IP, daily for 5 days). Mice were killed 2 mo after TM injections, and samples of long bones were prepared for frozen sections followed by the X-Gal staining. Eighty-four percent and 76% recombination efficiency was observed in 5- (A) and 8-mo-old (B) Col2a1-CreER^T2;R26R mice (n = 3), respectively, suggesting that floxed genes expressed in articular chondrocytes can be efficiently targeted at adult stage when they are crossed with Col2a1-CreER^T2 transgenic mice.

FIG. 2

Increased β-catenin protein levels in articular chondrocytes from β-catenin cAct mice. Three-month-old β-catenin cAct mice and Cre-negative control littermates were administered with TM (1 mg/10 g body weight, IP, daily for 5 days). Mice were killed 2 mo after TM injection, sections of long bones were prepared, and β-catenin immunostaining was performed. A significant increase in nuclear β-catenin levels were observed in β-catenin cAct mice.

FIG. 3

Five-month-old β-catenin cAct mice developed a mild OA-like phenotype. Three-month-old Col2a1-CreER^T2;β-catenin^fx(Ex3)/wt (β-catenin cAct) mice and Cre-negative control mice were administered TM (1 mg/10 g body weight, IP, daily for 5 days). Mice were killed 2 mo after TM induction. Histological sections were prepared, and Safranin O/Fast green and Alcian blue/hematoxylin & orange G staining was performed. Reduced Safranin O (A) and Alcian blue staining (B) (black arrows), loss of articular chondrocytes at the surface of knee joint (brown arrows), and destruction of articular cartilage (blue arrows) were observed in β-catenin cAct mice. Histomorphometric analysis showed that there was 38% reduction in the cartilage area in β-catenin cAct mice (n = 4) (C). *p < 0.05, unpaired Student's t-test.

FIG. 4

Eight-month-old β-catenin cAct mice developed a severe OA-like phenotype. Six-month-old Col2a1-Cre^T2;β-catenin^fx(Ex3)/wt (β-catenin cAct) mice and Cre-negative control mice were administered TM (1 mg/10 g body weight, IP, daily for 5 days). Mice were killed 2 mo after TM induction. Histological sections were prepared and Safranin O/Fast green and Alcian blue/hematoxylin & orange G staining was performed. Loss of the entire articular cartilage layer at the surface of knee joint (brown arrows), cell cloning (black arrows), and the formation of chondrophyte (blue arrows) were observed in β-catenin cAct mice (A-C). X-ray radiography showed that osteophyte formation in β-catenin cAct mice (red arrows) (D). High-magnification pictures of Safranin O/Fast green and Alcian blue/hematoxylin & orange G staining showed cell cloning (red arrows) (H), formation of clefts (black arrows) (J), loss of the entire articular cartilage layer (green arrows) (F and J), formation of chondrophyte (brown arrows; E and G-I), and new woven bone formation (blue arrow) (J) in knee joints from 8-mo-old Col2a1-CreER^T2;β-catenin^fx(Ex3)/wt (β-catenin cAct) mice.

FIG. 5

Chondrocyte differentiation is accelerated in β-catenin conditional activation (cAct) mice. Primary articular chondrocytes were isolated from β-catenin cAct mice and Cre-negative control mice (n = 10). Rounded cell morphology (A) and type I collagen (col1) and type II collagen (col2) expression (B) indicate that there is minimal fibroblast or osteoblast contamination in the cell culture. The expression of articular chondrocyte marker genes was analyzed by real-time RT-PCR. Among Bmp family members, Bmp2 is increased 6-fold. There are >2-fold increases in the expression of Bmp6 and Gdf5 (C). No change in Bmp4 expression was observed. The expression of aggrecan is increased 2.5-fold (D). The expression of two metalloproteases, Mmp-9 (4-fold) and Mmp-13 (3.5-fold), was also significantly increased (D). The mRNA levels of other chondrocyte maturation markers, such as Alp (2.5-fold), osteocalcin (Oc, 3-fold), and type X collagen (colX, 3.5-fold), were also significantly increased (E). To further confirm these results, we isolated articular tissues from 2-mo-old β-catenin cAct mice and Cre-negative control mice. Total RNA was extracted from these tissues and the expression of chondrocyte marker genes were examined by real-time RT-PCR. The results showed that the expression of colX (3-fold), Mmp-9 (2-fold), Mmp-13 (3-fold), and Oc (12-fold) was significantly increased in β-catenin cAct mice (F). Consistent with gene expression from isolated articular chondrocytes, the expression of Bmp2 (5-fold), but not Bmp4, was significantly increased in articular tissues derived from β-catenin cAct mice (G). *p < 0.05, unpaired Student's t-test. Six-month-old β-catenin cAct mice and Cre-negative control littermates were administered TM (1 mg/10 g body weight, IP, daily for 5 days). Mice were killed 2 mo after TM injections, sections of long bones were prepared, and MMP-13 immunostaining was performed. A significant increase in cellular MMP-13 protein levels was observed in β-catenin cAct mice (H).

FIG. 6

Activation of β-catenin signaling alters the expression of Wnt ligands, Wnt antagonist, and Wnt target gene. Primary articular chondrocytes were isolated from 1-mo-old β-catenin cAct mice and Cre-negative control mice (n = 8). The expression of canonical (Wnt1, Wnt3a, Wnt4, Wnt7a, and Wnt7b) and noncanonical (Wnt5 and Wnt11) Wnt ligands and Wnt antagonists (sFRP2 and WISP1) was analyzed by real-time RT-PCR. The expression of Wnt1, Wnt4, and Wnt7a was decreased 70–90% (A,B, and D), whereas no significant change was found on the expression of Wnt4 and Wnt7b (C and E). In contrast, the expression of Wnt5 and Wnt11 was increased 1.7- and 2.4-fold (F and G). In addition, the expression of sFRP2 and WISP1 was also increased (2.3- and 2.6-fold increase, respectively) (H and I).

FIG. 7

β-catenin levels were increased in human OA subjects. Normal human joints from trauma (n = 20) (A) and OA cartilage from knee arthroplasty patients (9 for low Mankin grade and 13 for high Mankin grade) (B and C) were fixed and processed for β-catenin immunostaining. β-catenin protein levels were significantly increased in mild (low Mankin-graded) and severe (high Mankin-graded) human OA knee joints (red arrows) (B and C).