SOX9 directly Regulates CTGF/CCN2 Transcription in Growth Plate Chondrocytes and in Nucleus Pulposus Cells of Intervertebral Disc.

Several lines of evidence indicate that connective tissue growth factor (CTGF/CCN2) stimulates chondrocyte proliferation and maturation. Given the fact that SOX9 is essential for several steps of the chondrocyte differentiation pathway, we asked whether Ctgf (Ccn2) is the direct target gene of SOX9. We found that Ctgf mRNA was down-regulated in primary sternal chondrocytes from Sox9(flox/flox) mice infected with Ad-CMV-Cre. We performed ChIP-on-chip assay using anti-SOX9 antibody, covering the Ctgf gene from 15 kb upstream of its 5'-end to 10 kb downstream of its 3'-end to determine SOX9 interaction site. One high-affinity interaction site was identified in the Ctgf proximal promoter by ChIP-on-chip assay. An important SOX9 regulatory element was found to be located in -70/-64 region of the Ctgf promoter. We found the same site for SOX9 binding to the Ctgf promoter in nucleus pulposus (NP) cells. The loss of Sox9 in growth plate chondrocytes in knee joint and in NP cells in intervertebral disc led to the decrease in CTGF expression. We suggest that Ctgf is the direct target gene of SOX9 in chondrocytes and NP cells. Our study establishes a strong link between two regulatory molecules that have a major role in cartilaginous tissues.

The postnatal role of Sox9 in cartilage.

Sox9 is an essential transcription factor for the differentiation of the chondrocytic lineage during embryonic development. To test whether Sox9 continues to play a critical role in cartilaginous tissues in the adult mice, we used an inducible, genetic strategy to disrupt the Sox9 gene postnatally in these tissues. The postnatal inactivation of Sox9 led to stunted growth characterized by decreased proliferation, increased cell death, and dedifferentiation of growth plate chondrocytes. Upon postnatal Sox9 inactivation in the articular cartilage, the sulfated proteoglycan and aggrecan content of the uncalcified cartilage were rapidly depleted and the degradation of aggrecan was accompanied by higher ADAMTS5 immunostaining and increased detection of the aggrecan neoepitope, NITEGE. In spite of the severe loss of Collagen 2a1 mRNA, the Collagen II protein persisted in the articular cartilage, and no histopathological signs of osteoarthritis were observed. The homeostasis of the intervertebral disk (IVD) was dramatically altered upon Sox9 depletion, resulting in disk compression and subsequent degeneration. Inactivation of Sox9 in the IVD markedly reduced the expression of several genes encoding extracellular matrix proteins, as well as some of the enzymes responsible for their posttranslational modification. Furthermore, the loss of Sox9 in the IVD decreased the expression of cytokines, cell-surface receptors, and ion channels, suggesting that Sox9 coordinates a large genetic program that is instrumental for the proper homeostasis of the cells contained in the IVD postnatally. Our results indicate that Sox9 has an essential role in the physiological control of cartilaginous tissues in adult mice. © 2012 American Society for Bone and Mineral Research.

Expression of master regulatory genes controlling skeletal development in benign cartilage and bone forming tumors.

Recent progress in skeletal molecular biology has led to the clarification of the transcriptional mechanisms of chondroblastic and osteoblastic lineage differentiation. Three master transcription factors-Sox9, Runx2, and Osterix-were shown to play an essential role in determining the skeletal progenitor cells' fate. The present study evaluates the expression of these factors in 4 types of benign bone tumors-chondromyxoid fibroma, chondroblastoma, osteoid osteoma, and osteoblastoma-using immunohistochemistry and tissue microarrays. Osteoid osteoma and osteoblastoma showed strong nuclear expression of Osterix and Runx2. In contrast, only a few chondroblastomas showed positive nuclear expression of Osterix. Strong nuclear expression of Sox9 was detected in all chondroblastomas, whereas nearly half of the osteoblastomas showed focal weak cytoplasmic expression of Sox9.

Generation of aggrecan-CreERT2 knockin mice for inducible Cre activity in adult cartilage.

The function of cartilage in the adult is dependent on a host of regulatory molecules such as growth factors, extracellular matrix, enzymes, signaling molecules, and transcription factors. However, germline mutations in some genes that are expressed in adult cartilage lead to embryonic or perinatal lethality. To examine the function of these and other genes postnatally, we have generated a targeted mouse by homologous recombination that "knocks in" the inducible Cre recombinase construct, CreERT2, in the 3' untranslated region of the endogenous mouse aggrecan gene (Agc1(tm(IRES-creERT2))). The properties and efficiency of the inducible cre recombinase were tested by examining X-gal staining of tissues from embryos as well as growing and adult Agc1(tm(IRES-creERT2)/+);Rosa 26R mice. These mice were injected with the inducer, tamoxifen, at different time points during embryonic development and postnatally up to 6 months of age. Strong X-gal staining was observed in growth plate and articular cartilage as well as the fibrocartilage of meniscus, trachea, and intervertebral discs reproducing the pattern of endogenous aggrecan gene expression. In conclusion, we have generated a mouse model in which genes implicated in cartilage degenerative diseases can be inactivated in a spatial and temporal fashion in postnatal and adult mice.