Lafont Jerome, Jacques Claire, Le Dreau Gwenvael, Calhabeu Frederico, Thibout Helene, Dubois Catherine, Berenbaum Francis, Laurent Maryvonne, Martinerie Cecile
INSERM U515, Hôpital Saint-Antoine, Paris Cedex 12, France.
J Bone Miner Res. 2005 Dec;20(12):2213-23. doi: 10.1359/JBMR.050818. Epub 2005 Aug 22.
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.
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.
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.
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.
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.
我们研究了功能尚不清楚的NOV/CCN3在软骨细胞分化中的作用。发现NOV可上调转化生长因子β2（TGF-beta2）和X型胶原蛋白，并在ATDC5细胞和原代软骨细胞中作为TGF-beta1的下游效应物发挥作用。因此，NOV是软骨形成的正向调节因子。
NOV/CCN3是一种属于CCN家族的基质细胞蛋白。越来越多的证据表明，NOV可能在细胞分化中发挥作用，尤其是在软骨形成过程中。在鸡胚发育过程中，NOV在软骨中的表达受到严格调控，并且在威尔姆斯瘤中，NOV的高表达与软骨分化相关。然而，NOV在软骨形成中的精确作用以及NOV的潜在靶基因尚不清楚。
用NOV重组蛋白处理ATDC5细胞和原代软骨细胞，或用NOV特异性小干扰RNA（siRNA）转染，通过定量逆转录聚合酶链反应（qRT-PCR）确定NOV对几种参与软骨细胞分化的分子表达的影响。还建立了表达NOV的稳定ATDC5克隆，以表明NOV是TGF-beta1的下游效应物。
我们确定，在软骨形成分化的早期阶段，ATDC5细胞中NOV/CCN3的表达增加，且早于TGF-beta2以及几种软骨细胞标志物如SRY（Y染色体性别决定区）-盒9（SOX9）或X型胶原蛋白的出现。当外源性给予时，NOV重组蛋白在ATDC5细胞和原代小鼠软骨细胞中均上调TGF-beta2和X型胶原蛋白的信使核糖核酸（mRNA）水平，但不影响SOX9的表达。这种调节在内源性水平也会发生，因为在原代软骨细胞中，NOV表达的下调与TGF-beta2和X型胶原蛋白的抑制相关。此外，我们发现当软骨细胞暴露于TGF-beta1去分化处理时，NOV表达下调，这进一步证明NOV可能抵消TGF-beta1对软骨细胞的影响。
本研究首次对参与软骨细胞分化的NOV的两个新靶点进行了表征，表明NOV在基因调控级联中与TGF-beta1共同发挥作用，并表明NOV是软骨形成的正向调节因子。
