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Keywords:

  • articular chondrocyte;
  • Cre-recombination;
  • β-catenin;
  • chondrocyte differentiation;
  • osteoarthritis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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/β-catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for β-catenin in this disease has not been reported. Because tissue-specific activation of the β-catenin gene (targeted by Col2a1-Cre) is embryonic lethal, we specifically activated the β-catenin gene in articular chondrocytes in adult mice by generating β-catenin conditional activation (cAct) mice through breeding of β-cateninfx(Ex3)/fx(Ex3) mice with Col2a1-CreERT2 transgenic mice. Deletion of exon 3 of the β-catenin gene results in the production of a stabilized fusion β-catenin protein that is resistant to phosphorylation by GSK-3β. In this study, tamoxifen was administered to the 3- and 6-mo-old Col2a1-CreERT2;β-cateninfx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of β-catenin protein was detected by immunostaining in articular cartilage tissues of β-catenin cAct mice. In 5-mo-old β-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 β-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 β-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 β-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 β-catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of β-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 β-catenin in the development of OA.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Osteoarthritis (OA) is a degenerative joint disease that mainly involves dysfunction of articular chondrocytes, the only cell type present in articular cartilage. Articular chondrocytes produce and maintain the extracellular matrix, which is responsible for providing the appropriate structure and function to cartilaginous tissue. The function of articular chondrocytes is regulated by a variety of growth factors, including Wnt family members. β-catenin is a key molecule in the canonical Wnt signaling pathway and plays a critical role in multiple steps during chondrocyte formation and maturation.

FrzB encodes the secreted frizzled-related protein 3 (sFRP3) that antagonizes the signaling of Wnt ligands through frizzled receptors.math image Recent human genetic association studies showed that substitution mutation of the Arg324Gly of sFRP3 blocks its ability to antagonize Wnt signaling and predisposes patients to OA in weight-bearing joints.math image Furthermore, Frzb KO mice are more susceptible to chemically induced OA.math image These findings suggest that activation of β-catenin signaling may be associated with the development of OA. To date, no direct genetic evidence has been reported.

Genetic evidence is critical for understanding the role of β-catenin in skeletal development. However, this is limited by the embryonic or immediate postnatal lethality of β-catenin gene deletion and activation. Somatic deletion of the β-catenin gene results in lethality at stage E7.5, which is before formation of the skeletal elements.math image Studies with conditional deletion of the β-catenin gene indicate that β-catenin plays a critical role in multiple steps during chondrogenesis and chondrocyte maturation. Conditional deletion of the β-catenin gene in early mesenchymal progenitor cells (targeted by Prx1-Cre or Dermo1-Cre) results in enhanced chondrogenesis,math image suggesting that β-catenin inhibits early mesenchymal cell differentiation into chondrocytes. Mice with conditional deletion of the β-catenin gene in proliferating chondrocytes (targeted by Col2a1-Cre) die at birth from respiratory distress caused by a cleft secondary palate and a small rib cage.math image The delayed development of hypertrophic chondrocytes and ossification in the radius, reduced chondrocyte proliferation, and delayed blood vessel invasion in the growth plates were observed in these conditional knockout (cKO) mice.math image Conditional activation of the β-catenin gene in chondrocytes (targeted by Col2a1-Cre), in which exon 3 of the β-catenin gene was flanked by the loxP sequence, results in embryonic lethality around E18.5 with markedly diminished cartilage formation.math image In E16.5 embryos, the growth plate is severely hypoplastic and disorganized with loss of the parallel columns of chondrocytes. Chondrocyte maturation is accelerated in these mice but occurs in the setting of severe chondrodysplasia.math image Exon 3 of the β-catenin gene encodes for the serine and threonine residues that are phosphorylated by GSK-3β and targeted by protein degradation mechanism.math image Overall, although conditional gene deletion and activation mutants in cartilage have provided insight regarding the role of β-catenin in early cartilage development, the role of β-catenin in articular cartilage function in the adult remains unknown.

Whereas human genetic association studies suggest that genetic factors make significant contributions to the pathogenesis of OA,math image the direct genetic evidence and molecular mechanism for OA pathogenesis are still largely unknown. Because OA normally occurs at adult patients and animals, the generation of a proper tool to specifically target adult articular chondrocytes is extremely important. Type II collagen is a chondrocyte-specific protein, and its expression is detected in growth plate and articular chondrocytes in long bones and other cartilaginous tissues in the body. The col2a1 promoter has been used in a variety of animal models to achieve tissue-specific gene expression in chondrocytes.math image In these studies, we generated transgenic mouse lines in which the Cre recombinase was fused to a mutated ligand binding domain of the human estrogen receptor (ER)math image driven by the col2a1 promoter (Col2a1-CreERT2). The fusion protein has been reported to be translocated into the nuclei in response to the estrogen antagonist tamoxifen (TM) or 4-hydroxy TM, an active metabolite of TM in vivo.math image This transgenic mouse model can serve as a valuable tool for gene targeting in a chondrocyte-specific and temporally controlled manner.math image

In this study, we bred Col2a1-CreERT2 transgenic mice with β-cateninfx(Ex3)/fx(Ex3) mice in which exon 3 of the β-catenin gene is floxed and created β-catenin conditional activation (cAct) mice. Changes in the expression of articular chondrocyte-specific genes and the structure and morphology of articular cartilage were examined in these mice. Our findings show acceleration of articular chondrocyte maturation and the development of the OA-like phenotype in β-catenin cAct mice. Our studies provided direct and definitive evidence about the critical role of β-catenin in maintaining normal function of articular chondrocytes and in the pathogenesis of OA.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Generation of transgenic mice

Col2a1-CreERT2 transgenic mice were bred with Rosa26 reporter mice. Methods for mouse genotyping including primer sequences are the same as described previously.math image TM (1 mg/10 g body weight/day, IP, daily for 5 days) was administered to the 3- and 6-mo-old mice that were killed 2 mo after TM induction at the age of 5 and 8 mo. Cre-recombination efficiency was evaluated by X-Gal staining. Nuclear Fast red staining was performed as a counterstain. β-cateninfx(Ex3)/fx(Ex3) mice were originally reported by Harada et al.math image and were provided to us by Dr Linheng Li (Stowers Institute for Medical Research, Kansas City, MO, USA). The sequences of PCR primers for genotyping β-cateninfx(Ex3)/fx(Ex3) mice are as follows: upper primer, 5′-AGGGTACCTGAAGCTCAGCG-3′ and lower primer, 5′-CAGTGGCTGACAGCAGCTTT-3′. The 412-bp PCR product was detected in wildtype (WT) mice, and the 645-bp PCR product was detected in homozygous β-cateninfx(Ex3)/fx(Ex3) mice. In heterozygous mice (β-cateninfx(Ex3)/wt), both 412- and 645-bp PCR products were detected. The Col2a1-CreERT2;β-cateninfx(Ex3)/w transgenic mice and their Cre-negative littermates were used as controls and were administered TM as the experimental animals for phenotype analysis and cellular function studies.

Histology and histomorphometry

Initial X-ray and histological analyses were performed. Knee joints from 5- and 8-mo-old Col2a1-CreERT2;β-cateninfx(Ex3)/wt transgenic mice and Cre-negative control mice were dissected, fixed in 10% formalin, decalcified, and embedded in paraffin. Serial midsagittal sections of knee joints were cut every 10 μm from both the medial and lateral compartments. The sections were stained with Alcian blue/hematoxylin & orange G (AB/H&OG) and Safranin O/Fast green (SO/FG). To quantify changes in articular cartilage area and articular chondrocyte numbers, articular cartilage was outlined on the tibial surface, and an area algorithm in the software ImagePro 4.5 (Leeds Precision Instruments, Minneapolis, MN, USA) was used to determine the pixel area of outlined articular cartilage from each section. Using this approach, the average articular cartilage area was determined from seven WT and β-catenin cAct knee joints.

Immunostaining

Tissue sections were deparaffinized by immersing in xylene, fixed with 4% paraformaldehyde for 15 min, and treated with 0.5% Triton for 15 min, followed by fixing with 4% paraformaldehyde for another 5 min. Sections were incubated with a rabbit anti-β-catenin polyclonal antibody (1:20 dilution; Cell Signaling, Danvers, MA, USA), goat anti-MMP-13 polyclonal antibody (1:100 dilution; Chemicon International, Temecula, CA, USA) overnight and then a horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min. Slides were mounted with Permount (Electron Microscopy Sciences, Hatfield, PA, USA) and visualized under a light microscope.

Cell isolation and cell culture

TM (1 mg/10 g body weight/d, IP, daily for 5 days) was administered into 1-mo-old Col2a1-CreERT2;β-cateninfx(Ex3)/wt transgenic mice and their Cre-negative littermates, which were killed 1 mo after TM induction (2 mo old). The mice were killed and genotyped using tail tissues obtained at death. The femoral articular cartilage caps were harvested, washed with PBS, and digested with 0.1% Pronase (Roche Applied Science, Indianapolis, IN, USA) in PBS and incubated for 30 min in a 37°C shaking water bath. This was followed by incubation in a solution of 0.1% collagenase A (Roche Applied Science) in serum-free DMEM for 4 h in a shaking water bath. The digestion solution was passed through 70-μm Swinnex filters to remove all residual fragments. The solution was centrifuged, and the cells were resuspended in complete medium (DMEM with 10% FBS and 1% penicillin/streptomycin). The media were changed every 3 days.

Total RNA extraction and real-time RT-PCR analysis

Total RNA extracted from primary articular chondrocytes or articular cartilage tissue was prepared using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. One microgram total RNA was used to synthesize cDNA by iScripts cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Primer names and sequences for real-time PCR are listed in Tables 1 and 2.

Table Table 1.. Primer Sequences for Marker Genes of Articular Chondrocytes
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Table Table 2.. Primer Sequences for Wnt Signaling Genes
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Human tissue procurement and fixation

For human tissue, an IRB-approved protocol was executed to collect discarded cartilage from orthopaedic surgery patients. Normal cartilage was collected from trauma/amputation patients, and arthritic cartilage was collected from patients undergoing total knee arthroplasty. All human samples were harvested without patient identifiers. After recovery, human tissue was fixed for between 2 and 10 days at room temperature in 10% neutral-buffered formalin. All samples were decalcified in a solution containing 10% wt/vol EDTA for 3 wk and embedded in paraffin. Embedded samples were cut with a microtome to generate 3-μm-thick sections that were mounted on positively charged slides, baked at 60°C for 30 min, deparaffinized in xylene, and rehydrated in decreasing concentrations of ethanol.

Mankin scoring in human tissue

Human tissue sections were stained with safranin O/fast green and graded using a modified version of the Mankin scale.math image Specifically, cartilage was assigned a grade as follows: 0 = normal cartilage; 1 = localized fibrillation; 2 = broadly distributed fibrillation; 3 = clefts to the transitional zone; 4 = clefts to radial zone; 5 = clefts to calcified cartilage; and 6 = complete disorganization. Two independent observers assigned grades to all samples studied and the distribution of averaged grades allowed for stratification of arthritic samples into two groups: low Mankin grade (mild/early OA, grade 1.7) and high Mankin grade (severe OA, grade 5.0). Expression of β-catenin protein was examined by immunohistochemical method.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

TM-induced Cre-recombination is achieved in adult Col2a1-CreERT2 transgenic mice

In previous studies, we showed the efficient Cre-recombination in articular chondrocytes after TM induction at early postnatal stages (TM was administered in 2-wk-old mice).math image In these studies, we determined whether TM-induced Cre-recombination can be achieved when the growth plate cartilage is fully developed. If so, we may be able to completely separate the role of a specific gene in articular chondrocytes from its potential effect on the growth plate cartilage, which may indirectly affect the function of articular cartilage. Col2a1-CreERT2 transgenic mice were bred with Rosa26 reporter mice.math image TM induction was performed in the 3- and 6-mo-old Col2a1-CreERT2;R26R mice. Mice were killed 2 mo after TM induction at the age of 5 and 8 mo, and Cre-recombination efficiency was evaluated by X-Gal staining.

Growth plate cartilage is fully developed when mice have reached 3 mo of age. When TM was administered in the 3-mo-old mice, an average of 84% (n = 3) Cre-recombination efficiency was achieved in articular chondrocytes 2 mo after TM induction as determined by X-Gal staining (Fig. 1A). Similar but slightly lower recombination efficiency (76%; n = 3) was achieved in articular chondrocytes when TM was administered in the 6-mo-old Col2a1-CreERT2;R26R mice followed by X-Gal staining 2 mo later (Fig. 1B). In contrast, <20% Cre-recombination efficiency was observed in growth plate chondrocytes in these mice (data not shown). These findings suggest that Col2a1-CreERT2 transgenic mice could be used to target the floxed β-catenin gene in articular chondrocytes specifically at the adult stage.

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Figure FIG. 1.. TM-induced Cre-recombination in adult articular chondrocytes. Three- and 6-mo-old Col2a1-CreERT2;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-CreERT2;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-CreERT2 transgenic mice.

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OA-like articular cartilage destruction was observed in β-catenin cAct mice

We bred Col2a1-CreERT2 transgenic mice with β-cateninfx(Ex3)/fx(Ex3) mice to generate Col2a1-CreERT2;β-cateninfx(Ex3)/wt (β-catenin cAct) mice. Because amino acids encoded by exon 3 contain critical GSK-3β phosphorylation sites, deletion of exon 3 of the β-catenin gene results in the production of a stabilized fusion protein, which is resistant to phosphorylation by GSK-3β.math image Three- and 6-mo-old β-catenin cAct mice and Cre-negative control mice were treated with TM. The mice were killed 2 mo after TM induction, and the increase of β-catenin protein levels in articular chondrocytes was detected in the 5-mo-old β-catenin cAct mice compared with their Cre-negative controls (Fig. 2).

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Figure 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.

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The articular cartilage phenotype of β-catenin cAct mice was analyzed by histology. Safranin O/Fast green and Alcian blue/hematoxylin & orange G staining was performed on 3-μm-thick formalin-fixed sections. Histological results showed that age-dependent progressive loss of the smooth surface of articular cartilage occurs in β-catenin cAct mice. At the age of 5 mo, mild degeneration was observed at the articular surface of knee joints. The Safranin O and Alcian blue staining was reduced, and articular chondrocytes were missing in the weight-bearing area of the articular surface in β-catenin cAct mice (Figs. 3A and 3B). Histomorphometric analysis showed that articular cartilage area was significantly reduced in β-catenin cAct mice (Fig. 3C). At 8 mo of age, severe destruction of articular cartilage was observed in β-catenin cAct mice. Cell cloning, surface fibrillation and vertical clefts, and formation of chondrophytes and osteophytes were observed (Figs. 4A–4J). Complete loss of articular cartilage layers and the formation of new woven bone in response to the loss of subchondral bone were also found in β-catenin cAct mice (Figs. 4A–4J). Histological analysis showed that eight of eight (100%) and seven of eight (87%) of the 5- and 8-mo-old β-catenin cAct mice, respectively, have articular cartilage destruction. In contrast, no articular cartilage damage was found in 5-mo-old Cre-negative control mice and only minor articular cartilage damage was found in one of eight of 8-mo-old Cre-negative control mice. Overall, these phenotypic changes resemble the clinical features commonly observed in OA patients.

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Figure FIG. 3.. Five-month-old β-catenin cAct mice developed a mild OA-like phenotype. Three-month-old Col2a1-CreERT2;β-cateninfx(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.

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Figure FIG. 4.. Eight-month-old β-catenin cAct mice developed a severe OA-like phenotype. Six-month-old Col2a1-CreT2;β-cateninfx(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-CreERT2;β-cateninfx(Ex3)/wt (β-catenin cAct) mice.

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Articular chondrocyte maturation is accelerated in β-catenin cAct mice

To determine changes in the maturation status of articular chondrocytes in β-catenin cAct mice, we isolated primary articular chondrocytes from 2-mo-old β-catenin cAct mice and Cre-negative control mice in which TM induction was performed at the age of 1 mo. Rounded cell morphology and expression of very low levels of type I collagen (col1) indicated that there was minimal fibroblast or osteoblast contamination of the primary articular chondrocyte cultures (Figs. 5A and 5B). The expression of articular chondrocyte marker genes was analyzed by real-time PCR. We first examined the expression of Bmp family members. Among them, Bmp2 was significantly upregulated (6-fold increase; Fig. 5C). There was a >2-fold increases in the expression of Bmp6 and Gdf5 (Fig. 5C). In contrast, Bmp4 expression was not changed (Fig. 5C). The expression of aggrecan was also increased 2.5-fold (Fig. 5D). The expression of two important matrix metalloproteases, Mmp-9 and Mmp-13, was also significantly increased (4- and 3.5-fold, respectively; Fig. 5D). The mRNA levels of other chondrocyte maturation markers, such as alkaline phosphatase (Alp) (2.5-fold), osteocalcin (Oc, 3-fold), and type X collagen (colX, 3.5-fold), were also significantly increased (Fig. 5E). To further confirm if articular chondrocyte maturation is accelerated in β-catenin cAct mice, we isolated articular tissues from the 1-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 was examined by real-time 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 (Fig. 5F). Consistent with gene expression from isolated articular chondrocytes, the expression of Bmp2, but not Bmp4, was significantly increased (5-fold) in articular tissues derived from β-catenin cAct mice (Fig. 5G). Immunostaining of sections from 8-mo-old β-catenin cAct and Cre-negative controls showed that MMP-13 protein levels are significantly increased in β-catenin cAct mice (Fig. 5H). Taken together, these findings clearly indicate that the chondrocyte maturation process is accelerated in β-catenin cAct mice.

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Figure 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).

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Alterations in expression of Wnt ligands, Wnt antagonist, and Wnt target gene

To determine whether conditional activation of the β-catenin gene causes changes in Wnt signaling, we analyzed changes in expression of Wnt ligands and antagonist and Wnt target gene that are involved in canonical and noncanonical Wnt signaling in articular chondrocytes. Primary articular chondrocytes were isolated from 1-mo-old β-catenin cAct mice and Cre-negative control mice in which TM induction was performed at the age of 2 wk. We found that expression of Wnt1, Wnt3a, and Wnt7a was significantly reduced (Figs. 6A, 6B, and 6D), whereas no significant changes were found in the expression of Wnt4 and Wnt7b (Figs. 6C and 6E) in articular chondrocytes derived from β-catenin cAct mice. In contrast, expression of Wnt5 and Wnt11 was significantly increased in articular chondrocytes in which β-catenin signaling is activated (Figs. 6F and 6G). In contrast to the Wnt ligands, expression of Wnt antagonist sFRP2, and Wnt target gene WISP1 was also significantly increased in articular chondrocytes derived from β-catenin cAct mice (Figs. 6H and 6I).

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Figure 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).

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β-Catenin levels are increased in human OA samples

We further determined if β-catenin signaling is activated in human OA samples using immunostaining methods. Articular cartilage samples from patients undergoing total knee arthroplasty (OA samples) and from trauma/amputation patients (negative controls) were processed for Mankin grading to determine severity of osteoarthritismath image and immunohistochemical analysis with an anti-β-catenin monoclonal antibody. The initial Mankin grading facilitated the stratification of OA samples into two groups: low Mankin grade (mild/early OA, average grade of 1.7, range 0–2.7) and high Mankin grade (severe OA, average grade of 5.0, range 3.3–8.7). Whereas the normal cartilage group showed no significant immunoreactivity with the β-catenin antibody (Fig. 7A), both the low and high Mankin-graded OA groups displayed a significant cellular β-catenin staining (Figs. 7B and 7C). Immunograding of all samples showed a significant upregulation of β-catenin in both the low and high Mankin groups compared with the normal control. These results establish a strong association between human OA and β-catenin expression.

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Figure 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).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Arthritis is the number one cause of disability in the United States. OA, the most common form of arthritis, is a noninflammatory degenerative joint disease characterized by dysfunction of articular chondrocytes, articular cartilage degradation, osteophyte formation, and subchondral sclerosis.math image OA affects nearly 21 million people in the United States. It is estimated that 80% of the population will have radiographic evidence of OA by age 65, although only 60% of those will be symptomatic.math image The progression of OA is slow and eventually results in destruction and total loss of articular cartilage of various joints, including fingers, knees, hips, and spine. The disease process leads to limitation of joint movement, joint deformity, joint stiffness, inflammation, and severe pain. Whereas there are several strategies to reduce symptoms and/or decelerate disease progression,math image there are few therapeutic approaches for OA patients. Treatments for OA include nonsteroidal anti-inflammatory drugs and local injections of glucocorticoid, and in severe cases, joint replacement surgery. Currently, there is limited information about the cellular and/or molecular events that occur during articular cartilage degeneration. Therefore, understanding these events would have a tremendous impact on the development of more effective therapeutic interventions.

A genetic contribution to OA has been suggested in several epidemiologic studies.math image Genome-wide scans, fine-scale mapping, and candidate gene association analyses have identified several loci that may be associated with hip OA.math image One such locus was identified by two separate genome-wide scans for familial OA susceptibility,math image and finer mapping suggested a peak linkage signal at the D2S2284 (2q31.1) region.math image Further single nucleotide polymorphism (SNP) analysis of eight candidate genes in this region showed an association of hip OA with a functional SNP of the Frzb gene.math image A haplotype coding for substitutions of two highly conserved arginine residues (Arg200Trp and Arg324Gly) in Frzb is a strong risk factor for primary hip OA with an OR of 4.1 (p = 0.004).math image A role for the same alleles/haplotypes in generalized radiographic OAmath image and in hip OAmath image has also been reported in other studies in white populations.

Frzb encodes secreted frizzled-related protein 3 (sFRP3) that antagonizes the signaling of Wnt ligands through frizzled receptors.math image It has been recently reported that the Arg324Gly substitution of sFRP3 is associated with the development of OA in affected patients.math image Furthermore, Frzb KO mice are more susceptible to chemically induced OA.math image These observations suggest that activation of β-catenin signaling may be associated with the development of OA, although no direct evidence has been reported. In this study, we showed for the first time that conditional activation of the β-catenin gene in articular chondrocytes in adult mice leads to OA-like articular cartilage destruction associated with accelerated chondrocyte differentiation, suggesting that β-catenin signaling plays a critical role in OA pathogenesis. This finding is consistent with reports from human genetic association studies in which patients with Frzb gene mutation (Arg324Gly substitution of the sFRP3 protein) have a higher frequency of OA occurrence. Because β-catenin cAct mice show spontaneous OA lesion in articular cartilage, it suggests that β-catenin may play a central role in OA development caused by Frzb mutations or other mechanisms that lead to activation of β-catenin signaling.

In this study, we found that mRNA expression of Bmp2 was significantly increased in articular chondrocytes and articular cartilage tissues (5- to 6-fold increase) derived from β-catenin cAct mice. Gene expression analysis also showed that expression of chondrocyte differentiation marker genes, regulated by BMP-2 such as Alp, Oc, and colX, were also significantly increased in articular chondrocytes derived from β-catenin cAct mice. It has been reported that BMP-2 induces de novo osteophyte formation in the normal murine knee joint.math image These observations suggest that β-catenin may regulate chondrocyte maturation and osteophyte formation in part through a Bmp2-dependent mechanism. In this study, we also found that the expression of Mmp-13 mRNA was increased in articular chondrocytes derived from β-catenin cAct mice. MMP-13 is a potent enzyme that degrades cartilage matrix with preference for type II collagen, and the expression of MMP-13 is upregulated in human OA knee joints.math image The transgenic mice expressing constitutively active Mmp-13 show changes in the OA-like phenotype,math image suggesting a close relationship between Mmp-13 and cartilage destruction in OA. In the future, we will further study the regulatory mechanisms of β-catenin with respect to the expression of Bmp2 and Mmp-13 in articular chondrocytes.

To determine changes in Wnt signaling, we examined expression of Wnt ligands and Wnt antagonist in articular chondrocytes in which β-catenin signaling is activated. It is known that Wnt1, Wnt3a, Wnt4, Wnt7a, and Wnt7b are involved in canonical Wnt signaling and Wnt5 and Wnt11 are involved in noncanonical Wnt signaling.math image In this study, we found that expression of Wnt1, Wnt3a, and Wnt7a was significantly reduced and expression of sFRP2 was significantly increased, suggesting that there is a negative feedback regulation in genes involved in canonical Wnt signaling. In contrast, expression of Wnt5 and Wnt11 was significantly increased, suggesting that activation of β-catenin signaling upregulates noncanonical Wnt signaling in articular chondrocytes.

The mechanism underlying β-catenin–induced OA is that β-catenin promotes articular chondrocyte maturation, which is consistent with the role of β-catenin in the developing growth plate. In Fig. 2, the β-catenin–positive cells in the resting zone have lost their flattened phenotype, suggesting that these cells are undergoing maturation as a result of increased β-catenin within the cells. In addition, β-catenin–positive cells are closer to the articular surface, possibly as a result of more efficient Cre-recombination caused by better tamoxifen penetration in superficial versus deep layers.

Recently, we found that selective inhibition of β-catenin signaling in chondrocytes causes delay of growth plate chondrocyte maturation and articular cartilage destruction in Col2a1-ICAT transgenic mice.math image We also found that cell apoptosis of articular chondrocytes is significantly increased in these transgenic mice.math image These findings suggest that β-catenin signaling plays a critical role in prevention of articular chondrocytes from undergoing apoptosis under normal physiological conditions.

In summary, in this study, we showed for the first time that conditional activation of the β-catenin gene in articular chondrocytes in adult mice leads to premature chondrocyte differentiation and the development of an OA-like phenotype. Our studies have provided novel and definitive evidence about the role of β-catenin signaling in articular chondrocyte function and OA pathogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by Grants R01 AR051189, R01 AR054465, K02 AR052411, and CORT Pilot (Supplement to P50 AR054041) to DC, R01 AR AR053717 to RO'K, and R01 AR045700 to RR from the National Institute of Health. The authors thank Kimberly A Napoli for assistance in preparing the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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