New target genes for NOV/CCN3 in chondrocytes: TGF-beta2 and type X collagen.
We studied the involvement of NOV/CCN3, whose function is poorly understood, in chondrocyte differentiation. NOV was found to upregulate TGF-beta2 and type X collagen and to act as a downstream effector of TGF-beta1 in ATDC5 and primary chondrocytes. Thus, NOV is a positive modulator of chondrogenesis.


BACKGROUND
NOV/CCN3 is a matricellular protein that belongs to the CCN family. A growing body of evidence indicates that NOV could play a role in cell differentiation, particularly in chondrogenesis. During chick embryo development, NOV expression is tightly regulated in cartilage, and a high expression of NOV has been associated with cartilage differentiation in Wilms' tumors. However, a precise role for NOV and potential target genes of NOV in chondrogenesis are unknown.


METHODS
ATDC5 cells and primary chondrocytes were either treated with NOV recombinant protein or transfected with a NOV-specific siRNA to determine, using quantitative RT-PCR, the effect of NOV on the expression of several molecules involved in chondrocyte differentiation. Stable ATDC5 clones expressing NOV were also established to show that NOV was a downstream effector of TGF-beta1.


RESULTS
We established that NOV/CCN3 expression increases in ATDC5 cells at early stages of chondrogenic differentiation and precedes the appearance of TGF-beta2 and of several chondrocytic markers such as SOX9 or type X collagen. When exogenously administered, NOV recombinant protein up-regulates TGF-beta2 and type X collagen mRNA levels both in ATDC5 cells and in primary mouse chondrocytes but does not influence SOX9 expression. This regulation also occurs at the endogenous level because downregulation of NOV expression is correlated with an inhibition of TGF-beta2 and type X collagen in primary chondrocytes. Furthermore, we found that NOV expression is downregulated when chondrocytes are exposed to TGF-beta1-dedifferentiating treatment in chondrocytes, further providing evidence that NOV may counteract TGF-beta1 effects on chondrocytes.


CONCLUSIONS
This study provides the first characterization of two new targets of NOV involved in chondrocyte differentiation, shows that NOV acts with TGF-beta1 in a cascade of gene regulation, and indicates that NOV is a positive modulator of chondrogenesis.
Lafont
Jerome
J
INSERM U515, Hôpital Saint-Antoine, Paris Cedex 12, France.
Jacques
Claire
C
Le Dreau
Gwenvael
G
Calhabeu
Frederico
F
Thibout
Helene
H
Dubois
Catherine
C
Berenbaum
Francis
F
Laurent
Maryvonne
M
Martinerie
Cecile
C
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics; D024121:Collagen Type X / Q000235:genetics*; D055513:Connective Tissue Growth Factor; D015870:Gene Expression / Q000187:drug effects; D006609:High Mobility Group Proteins / Q000235:genetics; D017874:Immediate-Early Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D007328:Insulin / Q000494:pharmacology; D039081:Integrin alpha5beta1 / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D051379:Mice; D055514:Nephroblastoma Overexpressed Protein; D011506:Proteins / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics*; D053781:Transforming Growth Factor beta2
C526318:CCN2 protein, mouse; C526320:Ccn3 protein, mouse; D024043:Collagen Type II; D024121:Collagen Type X; D006609:High Mobility Group Proteins; D017874:Immediate-Early Proteins; D007328:Insulin; D039081:Integrin alpha5beta1; D036341:Intercellular Signaling Peptides and Proteins; D055514:Nephroblastoma Overexpressed Protein; D011506:Proteins; D034741:RNA, Small Interfering; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D055513:Connective Tissue Growth Factor