CCN1 Regulates Chondrocyte Maturation and Cartilage Development

Abstract

WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis.

Keywords: CARTILAGE; CCN1; CHONDROCYTE; β-CATENIN.

Fig. 1

WNT/β-CATENIN signaling promotes CCN1 expression in chondrocytes. (A) Primary sternal chondrocytes from β-cateninfx(exon3)/fx(exon3) mice were infected with Ad5-CMV-GFP (Control) or Ad5-CMV-Cre (β-CAT GOF) and cultured for 6 days. Culture medium was then collected to detect CCN1 secretion using a membrane array spotted with antibodies to angiogenesis factors. Anti-CCN1 antibody spots from membranes incubated with Control or β-CAT GOF culture media are shown. (B) β-cateninfx(exon3)/fx(exon3) primary sternal chondrocytes were infected with Ad5-CMV-GFP (Control) or Ad5-CMV-Cre (β-CAT GOF) and harvested for mRNA isolation and quantitative real-time RT-PCR analyses of Ccn1 gene expression. p < 0.05, Student’s t test. (C) Wild-type primary sternal chondrocytes were treated with vehicle (Control) or WNT3A (100 ng/mL) for 48 hours and harvested for mRNA isolation and quantitative real-time RT-PCR analyses of Ccn1 gene expression. **p < 0.01, Student’s t test. (D) CCN1 immunohistochemistry of proximal humerus sections from E18.5 β-cateninfx(exon3)/+ (WT) and Col2a1-Cre+/-ERT2; β-cateninfx(exon3)/+ (β-CAT GOF) littermates administered tamoxifen at E13.5. Da and Db, 5× images; Dc–f, high-magnification images (20×) from the corresponding boxed regions in Da and Db. Arrows in Df point to ectopic epiphyseal CCN1 expression. (E) ATDC5 cells were transfected with 100 nM Control or β-catenin siRNA and then harvested 72 hours later for isolation of mRNA or protein. Quantitative real-time RT-PCR analyses was performed for analyses of Ccn1 gene expression. Western blotting was performed for the indicated proteins.**p < 0.01, Student’s t test. (F) CCN1 immunohistochemistry of proximal humerus sections from E18.5 β-cateninfx/fx (WT) and Col2a1-Cre+/-ERT2; β-cateninfx/fx (β-CAT LOF) littermates administered tamoxifen at E13.5. (F) CCN1 immunohistochemistry of knee joint sections from 2-month-old (P60) β-cateninfx(exon3)/+ (WT) and Acan-Cre+/-ERT2; β-cateninfx(exon3)/+ (β-CAT GOF) littermates administered tamoxifen at age 1 month. Fa and Fb, 5× images; Fc–f, high-magnification images (20×) from the corresponding boxed regions in Fa and Fb.

Fig. 2

CCN1 promotes chondrocyte maturation. (A) ATDC5 cells were cultured in chondrogenic differentiation media for the indicated number of days and then harvested for mRNA isolation and quantitative real-time RT-PCR analyses of Col2a1, Col10a1, Mmp13, and Ccn1 gene expressions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Fisher’s LSD multiple comparisons test. (B) ATDC5 cells were cultured in chondrogenic differentiation media for 0 or 8 days and then harvested for Western blotting of the indicated proteins. (C) ATDC5 cells were cultured in chondrogenic differentiation media and treated with control IgG or an anti-CCN1 neutralizing antibody. After 6 days, the cultures were stained with Alcian blue and Alizarin red or harvested for mRNA isolation and quantitative real-time RT-PCR analyses of Col2a1, Col10a1, and Mmp13 gene expressions. **p < 0.01, ***p < 0.001, Student’s t test. (D) ATDC5 cells were infected with Ad-Null or Ad-CCN1-V5 adenoviruses and cultured in chondrogenic differentiation media. After 6 days, the cultures were harvested for Western blotting, Alizarin red staining, or mRNA isolation and quantitative real-time RT-PCR analyses of Col2a1, Col10a1, and Mmp13 gene expressions. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test.

Fig. 3

CCN1 overexpression leads to chondrodysplasia and aberrant cartilage development. (A) Diagram of the conditional Ccn1 transgene. The CMV early enhancer/chicken b-actin promoter (CAG) drives Ccn1 expression from this transgene after removal of a loxP-flanked STOP cassette by Cre recombinase. Two independent transgenic mouse strains were generated and bred with Col2a1-Cre mice (designated Ccn1Tg-1 and Ccn1Tg-2). (B) Alcian blue/hematoxylin/Orange G staining of tibial growth plate sections from E18.5 Ccn1Tg-1 (Col2a1-Cre+/−; Tg+/−) and wild-type (WT) littermates (left panels) or P1 Ccn1Tg-2 (Col2a1-Cre+/−; Tg+/−) and wild-type (WT) littermates (right panels). Be-Bp are high-magnification images (40×) of the boxed regions in Ba–d. (C) Quantification of proliferating zone and hypertrophic zone cell number and size in E18.5 Col2a1-Cre; Ccn1Tg-1 embryos (Cre+, n = 5; WT, n = 4). (D) Quantification of proliferating zone and hypertrophic zone cell number and size in P1 Col2a1-Cre; Ccn1Tg-2 neonates (Cre+, n = 3; WT, n = 3). Values for the Tg mice were normalized to those of the WT mice and are presented as a fold change. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. RZ = resting zone; PZ = proliferating zone; HZ = hypertrophic zone.

Fig. 4

CCN1 overexpression leads to increased epiphyseal chondrocyte apoptosis. TUNEL staining of tibial growth plate sections from E18.5 Ccn1Tg-1 mice and WT littermates (a and b) or P1 Ccn1Tg-2 mice and WT littermates (c and d).

Fig. 5

CCN1 overexpression leads to postnatal cartilage degradation. (A) Alcian blue/hematoxylin/Orange G staining of knee joint sections from 2-month-old WT (Aa, Ac) and Ccn1Tg-2 (Ab, Ad) mice. Aa and Ab, 5× images; Ac and Ad, high-magnification images (20×) from the corresponding boxed regions in Aa and Ab. Articular cartilage degeneration/fibrosis on the distal femur (black arrows) and enlarged chondrocyte size (orange arrows) are indicated. (B) Alcian blue/hematoxylin/Orange G staining of knee joint sections from 6-month-old WT (Ba, Bc, Be) and Tg-2nd (Bb, Bd, Bf) mice. Ba and Bb, 5× images; Bc–f, high-magnification images (20×) of the articular and growth plate cartilages from the corresponding boxed regions in Ba and Bb. Articular cartilage degeneration/fibrosis on the distal femur (black arrow) is indicated. (C) MMP13 immunohistochemistry of knee joint sections from 6-month-old WT and Ccn1Tg-2 mice. Ca and Cb, nonspecific IgG primary antibody; Cc and Cd, anti-MMP13 primary antibody. (D) Quantification of articular chondrocyte numbers in 6-month-old Col2a1-Cre; Ccn1Tg-2 mice (Cre+, n = 3; WT, n = 4). Numbers for the Tg mice were normalized to those of the WT mice and are presented as a fold change. **p < 0.01, Student’s t test.

Fig. 6

Meniscal injury leads to elevated CCN1 expression in articular cartilage and synovium. (A) CCN1 immunohistochemistry of knee joint sections from mice subjected to sham or MLI at age 10 weeks. Joints were harvested 1 week post-injury. (B) Representative results of CCN1 immunohistochemistry on a tissue microarray of normal human cartilage or cartilage from patients undergoing arthroscopic surgery 4 weeks after meniscal injury.