Stephen P. Henry

Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.

One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.

Expression pattern and gene characterization of asporin. a newly discovered member of the leucine-rich repeat protein family

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression ofasporin partially overlaps with the expression ofdecorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22–9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2,osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.

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.

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 (Agc1tm(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 Agc1tm(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. genesis 47:805–814, 2009.

The Vitreous Glycoprotein Opticin Inhibits Preretinal Neovascularization

PURPOSE Opticin is an extracellular matrix glycoprotein that the authors discovered in the vitreous humor of the eye. It is synthesized by the nonpigmented ciliary epithelium and secreted into the vitreous cavity and, unusually for an extracellular matrix molecule, high-level synthesis is maintained into adult life. Here the authors investigated the hypothesis that opticin influences vascular development in the posterior segment of the eye and pathologic angiogenesis into the normally avascular, mature (secondary) vitreous. METHODS Opticin was localized in murine eyes by immunohistochemistry. An opticin knockout mouse was established and vascular development was compared between knockout and wild-type mice. Wild-type and opticin null mice were compared in the oxygen-induced retinopathy model, a model of pathologic angiogenesis, and this model was also used to assess the effects of intravitreal injection of recombinant opticin into eyes of wild-type mice. RESULTS Opticin colocalizes with the collagen type II-rich fibrillar network of the vitreous, the inner limiting lamina, the lens capsule, the trabecular meshwork, and the iris. Analyses of the hyaloid and retinal vasculature showed that opticin has no effect on hyaloid vascular regression or developmental retinal vascularization. However, using the oxygen-induced retinopathy model, the authors demonstrated that opticin knockout mice produce significantly more preretinal neovascularization than wild-type mice, and the intravitreal delivery of excess opticin inhibited the formation of neovessels in wild-type mice. CONCLUSIONS A lack of opticin does not influence vascular development, but opticin is antiangiogenic and inhibits preretinal neovascularization.

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 Sox9flox/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.