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. 2008 Nov;23(11):1751-64.
doi: 10.1359/jbmr.080615.

Cooperative regulation of chondrocyte differentiation by CCN2 and CCN3 shown by a comprehensive analysis of the CCN family proteins in cartilage

Harumi Kawaki  1 , Satoshi Kubota, Akiko Suzuki, Noureddine Lazar, Tomohiro Yamada, Tatsushi Matsumura, Toshihiro Ohgawara, Takeyasu Maeda, Bernard Perbal, Karen M Lyons, Masaharu Takigawa
Affiliations

Cooperative regulation of chondrocyte differentiation by CCN2 and CCN3 shown by a comprehensive analysis of the CCN family proteins in cartilage

Harumi Kawaki et al. J Bone Miner Res. 2008 Nov.

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 chondrocyte-associated 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.

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Figures

Figure FIG. 1.
Figure FIG. 1.
Histological examination and immunostaining for collagens and PCNA of WT and CCN2‐KO mice. (A and B) HE‐stained proximal end of tibias. (C and D) Masson Goldner–stained sections of tibias. (E and F) Safranin‐O–stained developing tibial growth plates. (G–N) Immunoreaction for type II and type X collagens and PCNA in the tibial growth plates. (O and P) Quantification of PCNA+ cells with values in the graphs expressed as percent positive cells and total number of cells in the same area. The cells in four adjacent sections (black boxes, 200 × 200‐μm area/section) were scored by an analyst blinded to the genotype. Data are the mean ± SE. *p < 0.05, **p < 0.001, significantly different from WT. Scale bar: 200 μm (A and B), 2 mm (C and D), and 100 μm (E–J).
Figure FIG. 2.
Figure FIG. 2.
Effect of the CCN2 deletion on the expression of Ccn family members and chondrocyte marker genes in vitro and immunohistochemical localization of CCN family proteins in vivo. (A) The mRNA expression profile of Ccn family members in WT chondrocytes at confluence. (B) The mRNA expression profile of Ccn family members in CCN2‐KO chondrocytes vs. WT ones. Data from CCN2‐KO were standardized against the respective expression levels in WT. (C) Comparison of mRNA levels of chondrocyte marker genes; Sox9, Aggrecan, Col2a1, and Col10a1. Data are means ± SE of eight samples from eight littermates. (D) Western immunoblot analysis of the CCN proteins in WT vs. CCN2‐KO chondrocyte cell lysates. (E) Immunolocalization of each CCN member at E14.5. Negative control (NC) sections were incubated with 5 μg/ml of normal rabbit and goat antibodies. (F–I) The mRNA expression profile of Ccn3 and chondrocyte marker genes in CCN2‐KO cartilage from embryonic rib cage in comparison with that of WT. Data are means ± SE of four samples from each of four litter mates. *p < 0.05, significantly different from WT.
Figure FIG. 3.
Figure FIG. 3.
Time course of calcification and differential expression of Ccn family members and chondrocyte marker genes during terminal differentiation of WT and CCN2‐KO chondrocytes in vitro. (A) Phase‐contrast views at the initial confluence (day 7) and culture dishes showing mineral deposition by both types of chondrocytes during long‐term culture. (B–J) The gene‐expression profile during terminal differentiation. Data are mean ± SD of three independent determinations of triplicate examinations. Samples were collected from four WT and four CCN2‐KO mice from four litter mates for each type.
Figure FIG. 4.
Figure FIG. 4.
Effect of exogenous rCCN2 and rCCN3 on the proliferation, proteoglycan synthesis/maturation, and calcification of chondrocytes. (A and B) A comparative analysis of effects of rCCN2 and rCCN3 on the proliferation and proteoglycan synthesis between CCN2‐KO and WT chondrocytes. (C) Effects of rCCN2 and rCCN3 on the morphology, ECM accumulation, and mineral deposition of WT chondrocytes was evaluated. (D and E) A quantitative data of C is shown in graphic form. Data are the mean ± SE of two independent experiments. Chondrocytes were harvested from eight WT and eight CCN2‐KO mice. *p < 0.05, significantly different from WT control. † p < 0.05, significantly different from CCN2‐KO control.
Figure FIG. 5.
Figure FIG. 5.
Effect of exogenous CCN2 and rCCN3 on the gene expression of their own and other chondrocyte differentiation‐associated genes. (A–C) Expression of Ccn2 and Ccn3. (D–K) Expression of chondrocyte marker genes. Changes in mRNA expression levels of Ccn2 (A), Ccn3 (B and C), Aggrecan (D and E), Sox9 (F and G), Col2a1 (H and I), and Col10a1 (J and K) in WT (WT in parentheses: A, B, D, F, H, J) and CCN2‐KO (KO in parentheses: C, E, G, I, K) chondrocytes after addition of rCCN2 or rCCN3 were evaluated. All of the data were standardized against the value of the control at each time point. Data are the mean ± SD of four independent sets of duplicate samples from eight WT and eight CCN2‐KO mice.
Figure FIG. 6.
Figure FIG. 6.
Immunohistochemical and in vitro gene expression analysis of PTHrP and Ihh in CCN2‐KO vs. WT mice. (A–F) E17.5 tibias from WT and MT mice immunostained with anti‐PTHrP, anti‐PTH/PTHrP receptor, or anti‐Ihh antibody. (G and H) Pthrp and Ihh mRNA expression levels in WT and CCN2‐KO chondrocytes during terminal differentiation evaluated from the samples used in Figs. 3B–3J. (I–L) Effects of rCCN2 or rCCN3 on the mRNA level of Pthrp (I and J) and Ihh (K and L) in WT and CCN2‐KO chondrocytes evaluated by using the samples used for Fig. 5. Data are the mean ± SD of four independent sets of duplicate samples from eight WT and eight CCN2‐KO mice.
Figure FIG. 7.
Figure FIG. 7.
Mechanism of action of CCN2 and CCN3 in regulatory endochondral bone formation. (A–H) Effect of Ccn3 knockdown on the expression of chondrocyte‐associated genes in WT and CCN2‐KO chondrocytes in culture. Data are the mean ± SE of four independent sets of duplicate samples from four WT and four CCN2‐KO mice. *p < 0.05, **p < 0.01, significantly different from WT control. † p < 0.05, †† p < 0.01, significantly different from CCN2‐KO control. (I) Possible scheme of the cooperative regulation of chondrocyte differentiation by the counteracting functions of CCN2 and CCN3 under interaction with the PTHrP‐Ihh network. Dotted lines indicate the hypothetical pathways.
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