CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage

Abstract

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H₂O₂ caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.

Keywords: CCN3; NOV; SASP; aging; cellular communication network factor 3; oxidative stress; p21; p53; primary chondrocytes; senescence.

Figure 1

Comparison of mRNA expression levels of (A–C) Ccn3, (D) p16, (E) p53, (F) p21, (G) Acan, (H) Col2a1, (I,J) Sox9, (K) Il-6, and (L) Il-8 in articular chondrocytes of different ages (embryo E18.5 represents RNA from ribcage chondrocytes of embryonic day 18.5). The expression level of Ccn3 mRNA was standardized with (A) Gapdh, (B) Tmem199, a gene with little fluctuation in expression during aging, and (C) RNA amount. The expression level of Acan mRNA dramatically decreased with age, but Col2a1 mRNA expression did not. Sox9 mRNA expression increased when it was standardized by Gapdh; however, the expression level/μg RNA decreased with age. (K,L) Il-6 and Il-8, as senescence-associated secretory phenotype (SASP) factors, also showed increased expression with age (p < 0.05). Either rib cage (E.18.5) or knee articular (postnatal) cartilage was pooled from 8 embryos or 4 postnatal animals and seeded into at least 3 dishes. RNA from each dish was analyzed with triplicate by real time PCR, and the average of the value from one RNA sample was plotted as one ‘dot’. (M–P) Immunohistochemical staining of CCN3 in one-, two-, and seven-month old mouse articular cartilage. CCN3-positive chondrocytes (arrow) increased in the superficial layer of articular cartilage (dotted circle) of seven-month-old compared with one-month-old mice. No signal was observed without CCN3-specific antibody (Figure 1P, -1st Ab). Scale bar: 200 µm.

Figure 2

Increased expression of Ccn3 mRNA and protein in H₂O₂-treated chondrocytes. (A) Human primary articular chondrocytes isolated from 48 years old patient and (B) RCS cells were treated with increased concentration of H₂O₂ as indicated for 2 h to induce artificial senescence by oxidative stress, followed by changing to the normal growing media without H₂O₂. Total RNA was collected 24 h after changing media, and (A) CCN3, p21, p53, COL2A1, ACAN, COL10A1, MMP13 mRNA, and (B) Ccn3, p21, p53, Col2a1, Acan, Col10a1, Mmp13, and Adamts5 mRNA levels were monitored and standardized with Gapdh. The data represent the mean ± SD (n = 3 individual cultures); * p ≤ 0.05. (C) Cell lysates were collected both 24 and 48 h after changing media and CCN3 protein level was monitored with an anti-CCN3 antibody. (D, left) Cell lysates were collected 48 h after changing media and p53 protein level was monitored with an anti-p53 antibody. (D, right) Band intensity of p53 was standardized to βactin. (E) Colorimetric detection of senescence associated β galactosidase in H₂O₂-treated RCS cells. RCS cells were treated with H₂O₂ at indicated concentration for 2 h and after media change further incubated for 36 h. bar: 200 μm.

Figure 2

Increased expression of Ccn3 mRNA and protein in H₂O₂-treated chondrocytes. (A) Human primary articular chondrocytes isolated from 48 years old patient and (B) RCS cells were treated with increased concentration of H₂O₂ as indicated for 2 h to induce artificial senescence by oxidative stress, followed by changing to the normal growing media without H₂O₂. Total RNA was collected 24 h after changing media, and (A) CCN3, p21, p53, COL2A1, ACAN, COL10A1, MMP13 mRNA, and (B) Ccn3, p21, p53, Col2a1, Acan, Col10a1, Mmp13, and Adamts5 mRNA levels were monitored and standardized with Gapdh. The data represent the mean ± SD (n = 3 individual cultures); * p ≤ 0.05. (C) Cell lysates were collected both 24 and 48 h after changing media and CCN3 protein level was monitored with an anti-CCN3 antibody. (D, left) Cell lysates were collected 48 h after changing media and p53 protein level was monitored with an anti-p53 antibody. (D, right) Band intensity of p53 was standardized to βactin. (E) Colorimetric detection of senescence associated β galactosidase in H₂O₂-treated RCS cells. RCS cells were treated with H₂O₂ at indicated concentration for 2 h and after media change further incubated for 36 h. bar: 200 μm.

Figure 3

Induction of p21 promoter activity by overexpression of CCN3 in RCS cells. Detection of GFP-CCN3 (▶) and GFP-2Flag-CCN3 (▷) overexpression with (A) an anti-GFP antibody, and (B) anti-CCN3 antibody. Two days after transfection of pEGFP-CCN3 and pEGFP-2Flag-CCN3 vector (2 clones each) by electroporation, proteins were extracted from the cells and subjected to Western blot analysis. For the p21 promoter assay in (C), we used pEGFP-CCN3. (C) pEGFP-CCN3 vector, pGL3-PG12S containing p21 promoter with 12× multiple p53-binding sites, and pGL3-Renilla vector were transfected by electroporation, and two days after transfection, firefly luciferase activity was monitored and standardized with Renilla luciferase activity (The data represent the mean ± SD (n = 3 individual cultures); * p < 0.05).

Figure 4

Stimulation of p21 and p53 mRNA expression by recombinant CCN3 treatment in (A) mouse primary articular chondrocytes from 2w knee joints and (B) RCS cells. Recombinant CCN3 was added to the media at indicated concentrations (A) and 100 µg/mL (B). Total RNA was harvested after 24 h incubation. The data represent the mean ± SD (n = 5 individual cultures). * p < 0.05, ** p < 0.01.

Figure 5

Degradative changes in knee joints and rib chondrocytes as a result of cartilage specific CCN3 overexpression in mice. (A) Whole mount LacZ staining of transgenic knee joints harboring a Col2a1 promoter-GFP-CCN3-LacZ construct (medial view). Beta-galactosidase activity was traceable up to 18 months after birth. (B) Histological analysis of (A) and counterstained with eosin. (C,D) Safranin-O staining of two-month-old (C) and seven-month-old (D) Ccn3^tg mice. (E) Immunohistochemical staining of Aggrecan Neoepitope in two-month-old Ccn3^tg mice knee articular cartilage. (wt -1st Ab) Negative control without 1st antibody of wt sections. (F,G) Analysis of gene expression of extracellular matrices and matrix metalloproteinases in chondrocyte RNA obtained from (F) rib cage chondrocytes of embryonic 18.5-day-old and (G) ribcages of three-month-old of tg mice. (F) Increased expression of p53, p21 and decreased expression Col2a1 mRNAs, were observed. (G) High expression levels of p21 and decreased expression levels of Col2a1, Acan, and Sox9 were observed. Furthermore, the expression levels of inflammatory factors Il6, Il8, and Tnfα were increased. The p53 expression did not show significant differences (data not shown). Scale bar: 200 µm.

Figure 5

Degradative changes in knee joints and rib chondrocytes as a result of cartilage specific CCN3 overexpression in mice. (A) Whole mount LacZ staining of transgenic knee joints harboring a Col2a1 promoter-GFP-CCN3-LacZ construct (medial view). Beta-galactosidase activity was traceable up to 18 months after birth. (B) Histological analysis of (A) and counterstained with eosin. (C,D) Safranin-O staining of two-month-old (C) and seven-month-old (D) Ccn3^tg mice. (E) Immunohistochemical staining of Aggrecan Neoepitope in two-month-old Ccn3^tg mice knee articular cartilage. (wt -1st Ab) Negative control without 1st antibody of wt sections. (F,G) Analysis of gene expression of extracellular matrices and matrix metalloproteinases in chondrocyte RNA obtained from (F) rib cage chondrocytes of embryonic 18.5-day-old and (G) ribcages of three-month-old of tg mice. (F) Increased expression of p53, p21 and decreased expression Col2a1 mRNAs, were observed. (G) High expression levels of p21 and decreased expression levels of Col2a1, Acan, and Sox9 were observed. Furthermore, the expression levels of inflammatory factors Il6, Il8, and Tnfα were increased. The p53 expression did not show significant differences (data not shown). Scale bar: 200 µm.

Figure 6

Elevated expression of Ccn3, p53, and p21 mRNA in aged human articular cartilage. (A) Comparison of mRNA expression levels of CCN3, p53, and p21 in the primary culture of human articular chondrocytes at different ages. The expression level of Ccn3, p53, and p21 mRNA was standardized to Gapdh. From one patient, articular cartilage was collected and digested, and cultured in multiple dishes. Total RNA was collected at least 3 dishes individually, and real time-PCR was performed in triplicates for each RNA sample; one ‘dot’ indicates average of gene expression level from one patient. (positive correlation, all p < 0.01). (B) Western blot analysis of CCN3 protein in primary culture of the articular chondrocytes (8 μg protein/lane) from different ages (y) as indicated. (C) Immunohistochemical staining of CCN3 in human articular cartilage (tibiae) of several ages. Top: lower magnification; bottom: higher magnification of the black square in the top. Scale bar: 200 µm.