Abstract
To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.
Highlights
CCN2/CTGF (CCN family member 2/connective tissue growth factor, CCN2) is a cartilage-maintaining protein that is dominantly expressed in cartilage; and it strongly enhances the production of cartilaginous matrix proteins, such as type II collagen (Col2a1) and aggrecan, as well as stimulates chondrocyte proliferation, differentiation and maturation of growth-plate chondrocytes under physiological conditions [1,2,3,4,5]
The immunohistochemical analysis revealed significantly enhanced accumulation of CCN2 protein in growth-plate cartilage in the superficial and deep zones of articular cartilage of knee joints from 21-month-old transgenic mice (TG) mice (Figure 1E, Figure S1F); the number of X-gal-positive cells was lower in 5-monthold mice (Figure 1D), suggesting that overexpressed CCN2 had stably accumulated in the extracellular matrices
We evaluated the in vivo effect of cartilage-specific overexpression of CCN2 on the maintenance of knee joint cartilage in aging CCN2-transgenic mice
Summary
CCN2/CTGF (CCN family member 2/connective tissue growth factor, CCN2) is a cartilage-maintaining protein that is dominantly expressed in cartilage; and it strongly enhances the production of cartilaginous matrix proteins, such as type II collagen (Col2a1) and aggrecan, as well as stimulates chondrocyte proliferation, differentiation and maturation of growth-plate chondrocytes under physiological conditions [1,2,3,4,5]. Implantation of CCN2-incorporated gelatin hydrogel into full-thickness defects of rat articular cartilage accelerate cartilage repair [11] This raises the question as to whether, and if so, to what extent CCN2 is involved in the maintenance of the chondrocyte phenotype and protection from degenerative changes in aging cartilage or in osteoarthritis (OA). The disease is characterized by breakdown of the cartilage matrix followed by development of surface fibrillations and fissures, and these changes can lead to complete loss of articular cartilage. Another characteristic of OA is hypertrophy and ectopic growth of bony structures in the joints. Thickening of the subchondral bone combined with loss of articular cartilage leads to increased stiffness and reduced shock-absorbing capacity of the bone [14,15]
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