Cooperative Regulation of Chondrocyte Differentiation by CCN2 and CCN3 Shown by a Comprehensive Analysis of the CCN Family Proteins in Cartilage

Abstract

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and its null mice display skeletal dysmorphisms. However, little is known concerning roles of the other CCN members in chondrocytes. Using both in vivo and in vitro approaches, we conducted a comparative analysis of CCN2-null and wildtype mice to study the roles of CCN2 and the other CCN proteins in cartilage development. Immunohistochemistry was used to evaluate the localization of CCN proteins and other chondrocyte-associated molecules in the two types of mice. Moreover, gene expression levels and the effects of exogenous CCN proteins on chondrocyte proliferation, differentiation, and the expression of chondrocyteassociated genes in their primary chondrocytes were evaluated. Ccn3 was dramatically upregulated in CCN2-null cartilage and chondrocytes. This upregulation was associated with diminished cell proliferation and delayed differentiation. Consistent with the in vivo findings, CCN2 deletion entirely retarded chondrocyte terminal differentiation and decreased the expression of several chondrocyte-associated genes in vitro, whereas Ccn3 expression drastically increased. In contrast, the addition of exogenous CCN2 promoted differentiation strongly and induced the expression of the associated genes, whereas decreasing the Ccn3 expression. These findings collectively indicate that CCN2 induces chondrocyte differentiation by regulating the expression of chondrocyte-associated genes but that these effects are counteracted by CCN3. The lack of CCN2 caused upregulation of CCN3 in CCN2-null mice, which resulted in the observed phenotypes, such as the resultant delay of terminal differentiation. The involvement of the PTHrP-Ihh loop in the regulation of CCN3 expression is also suggested.

Spatial-temporal modulation of CCN proteins during wound healing in human skin in vivo

“…It is interesting to note that, as discussed above for CCN5, a similar CCN2-antagonizing function has been described for CCN3 in murine cartilage (Kawaki et al 2008) and in kidney mesangial cells (Riser et al 2009). Additional experiments will be needed to determine if CCN3 and CCN5 inhibit CCN2 function similarly but at different locations, or if their inhibitory activity is mechanistically different due to their intrinsic structural differences (CCN5 is the only CCN family member that lacks the entire C-terminal module).…”

CCN3-mediated promotion of sulfated proteoglycan synthesis in rat chondrocytes from developing joint heads

“…Note that ccn3 mRNA showed even remarkably higher levels of expression than the housekeeping (gapdh) or articular chondrocyte marker (tenascin C: tnc) gene (Kawaki et al 2008b), which is a typical phenotypic marker of differentiated chondrocytes, in mouse costal chondrocytes is rather repressed by rCCN3. In order to examine whether CCN3 exerts a similar, or different, effect on the chondrocytes from the joint head, we comparatively evaluated the uptake of radiolabeled sulfate by those cells in the absence or presence of rCCN3 at different doses.…”

WNT1-induced Secreted Protein-1 (WISP1), a Novel Regulator of Bone Turnover and Wnt Signaling

“…Loss-of-function studies in mice (12,13) showed that CCN2 is needed for proper bone development during embryogenesis. In mature mice, however, a different paradigm for CCN2 function emerges.…”