CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

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.     

Preservation of the chondrocyte's pericellular matrix improves cell‐induced cartilage formation

The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real‐time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross‐linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>50‐fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell‐induced cartilage production. J. Cell. Biochem. 110: 260–271, 2010. © 2010 Wiley‐Liss, Inc.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.