The role of CCN2 in cartilage and bone development

CCN2, a classical member of the CCN family of matricellular proteins, is a key molecule that conducts cartilage development in a harmonized manner through novel molecular actions. During vertebrate development, all cartilage is primarily formed by a process of mesenchymal condensation, while CCN2 is induced to promote this process. Afterwards, cartilage develops into several subtypes with different fates and missions, in which CCN2 plays its proper roles according to the corresponding microenvironments. The history of CCN2 in cartilage and bone began with its re-discovery in the growth cartilage in long bones, which determines the skeletal size through the process of endochondral ossification. CCN2 promotes physiological developmental processes not only in the growth cartilage but also in the other types of cartilages, i.e., Meckel’s cartilage representing temporary cartilage without autocalcification, articular cartilage representing hyaline cartilage with physical stiffness, and auricular cartilage representing elastic cartilage. Together with its significant role in intramembranous ossification, CCN2 is regarded as a conductor of skeletogenesis. During cartilage development, the CCN2 gene is dynamically regulated to yield stage-specific production of CCN2 proteins at both transcriptional and post-transcriptional levels. New functional aspects of known biomolecules have been uncovered during the course of investigating these regulatory systems in chondrocytes. Since CCN2 promotes integrated regeneration as well as generation (=development) of these tissues, its utility in regenerative therapy targeting chondrocytes and osteoblasts is indicated, as has already been supported by experimental evidence obtained in vivo

CCN3 modulates bone turnover and is a novel regulator of skeletal metastasis

The CCN family of proteins is composed of six secreted proteins (CCN1-6), which are grouped together based on their structural similarity. These matricellular proteins are involved in a large spectrum of biological processes, ranging from development to disease. In this review, we focus on CCN3, a founding member of this family, and its role in regulating cells within the bone microenvironment. CCN3 impairs normal osteoblast differentiation through multiple mechanisms, which include the neutralization of pro-osteoblastogenic stimuli such as BMP and Wnt family signals or the activation of pathways that suppress osteoblastogenesis, such as Notch. In contrast, CCN3 is known to promote chondrocyte differentiation. Given these functions, it is not surprising that CCN3 has been implicated in the progression of primary bone cancers such as osteosarcoma, Ewing’s sarcoma and chondrosarcoma. More recently, emerging evidence suggests that CCN3 may also influence the ability of metastatic cancers to colonize and grow in bone

Connective tissue growth factor is required for skeletal development and postnatal skeletal homeostasis in male mice.

Connective tissue growth factor (CTGF), a member of the cysteine-rich 61 (Cyr 61), CTGF, nephroblastoma overexpressed (NOV) (CCN) family of proteins, is synthesized by osteoblasts, and its overexpression inhibits osteoblastogenesis and causes osteopenia. The global inactivation of Ctgf leads to defective endochondral bone formation and perinatal lethality; therefore, the consequences of Ctgf inactivation on the postnatal skeleton are not known. To study the function of CTGF, we generated Ctgf(+/LacZ) heterozygous null mice and tissue-specific null Ctgf mice by mating Ctgf conditional mice, where Ctgf is flanked by lox sequences with mice expressing the Cre recombinase under the control of the paired-related homeobox gene 1 (Prx1) enhancer (Prx1-Cre) or the osteocalcin promoter (Oc-Cre). Ctgf(+/LacZ) heterozygous mice exhibited transient osteopenia at 1 month of age secondary to decreased trabecular number. A similar osteopenic phenotype was observed in 1-month-old Ctgf conditional null male mice generated with Prx1-Cre, suggesting that the decreased trabecular number was secondary to impaired endochondral bone formation. In contrast, when the conditional deletion of Ctgf was achieved by Oc-Cre, an osteopenic phenotype was observed only in 6-month-old male mice. Osteoblast and osteoclast number, bone formation, and eroded surface were not affected in Ctgf heterozygous or conditional null mice. In conclusion, CTGF is necessary for normal skeletal development but to a lesser extent for postnatal skeletal homeostasis

Connective tissue growth factor is required for skeletal development and postnatal skeletal homeostasis in male mice.

Connective tissue growth factor (CTGF), a member of the cysteine-rich 61 (Cyr 61), CTGF, nephroblastoma overexpressed (NOV) (CCN) family of proteins, is synthesized by osteoblasts, and its overexpression inhibits osteoblastogenesis and causes osteopenia. The global inactivation of Ctgf leads to defective endochondral bone formation and perinatal lethality; therefore, the consequences of Ctgf inactivation on the postnatal skeleton are not known. To study the function of CTGF, we generated Ctgf(+/LacZ) heterozygous null mice and tissue-specific null Ctgf mice by mating Ctgf conditional mice, where Ctgf is flanked by lox sequences with mice expressing the Cre recombinase under the control of the paired-related homeobox gene 1 (Prx1) enhancer (Prx1-Cre) or the osteocalcin promoter (Oc-Cre). Ctgf(+/LacZ) heterozygous mice exhibited transient osteopenia at 1 month of age secondary to decreased trabecular number. A similar osteopenic phenotype was observed in 1-month-old Ctgf conditional null male mice generated with Prx1-Cre, suggesting that the decreased trabecular number was secondary to impaired endochondral bone formation. In contrast, when the conditional deletion of Ctgf was achieved by Oc-Cre, an osteopenic phenotype was observed only in 6-month-old male mice. Osteoblast and osteoclast number, bone formation, and eroded surface were not affected in Ctgf heterozygous or conditional null mice. In conclusion, CTGF is necessary for normal skeletal development but to a lesser extent for postnatal skeletal homeostasis

Nephroblastoma overexpressed (Nov) inactivation sensitizes osteoblasts to bone morphogenetic protein-2, but nov is dispensable for skeletal homeostasis.

Overexpression of nephroblastoma overexpressed (Nov), a member of the Cyr 61, connective tissue growth factor, Nov family of proteins, inhibits osteoblastogenesis and causes osteopenia. The consequences of Nov inactivation on osteoblastogenesis and the postnatal skeleton are not known. To study the function of Nov, we inactivated Nov by homologous recombination. Nov null mice were maintained in a C57BL/6 genetic background after the removal of the neomycin selection cassette and compared with wild-type controls of identical genetic composition. Nov null mice were identified by genotyping and absent Nov mRNA in calvarial extracts and osteoblast cultures. Nov null mice did not exhibit developmental skeletal abnormalities or postnatal changes in weight, femoral length, body fat, or bone mineral density and appeared normal. Bone volume and trabecular number were decreased only in 1-month-old female mice. In older mice, after 7 months of age, osteoblast surface and bone formation were increased in females, and osteoclast and eroded surfaces were increased in male Nov null mice. Calvarial osteoblasts from Nov null mice displayed enhanced alkaline phosphatase activity, alkaline phosphatase mRNA, and transactivation of a bone morphogenetic protein (BMP)/phosphorylated mothers against decapentaplegic reporter construct in response to BMP-2. Similar results were obtained after the down-regulation of Nov by RNA interference in ST-2 stromal and MC3T3 cells. Osteoclast number was increased in marrow stromal cell cultures from Nov null mice. Surface plasmon resonance demonstrated direct interactions between Nov and BMP-2. In conclusion, Nov sensitizes osteoblasts to BMP-2, but Nov is dispensable for the maintenance of bone mass

Integrative approaches to the prediction of protein functions based on the feature selection

<p>Abstract</p> <p>Background</p> <p>Protein function prediction has been one of the most important issues in functional genomics. With the current availability of various genomic data sets, many researchers have attempted to develop integration models that combine all available genomic data for protein function prediction. These efforts have resulted in the improvement of prediction quality and the extension of prediction coverage. However, it has also been observed that integrating more data sources does not always increase the prediction quality. Therefore, selecting data sources that highly contribute to the protein function prediction has become an important issue.</p> <p>Results</p> <p>We present systematic feature selection methods that assess the contribution of genome-wide data sets to predict protein functions and then investigate the relationship between genomic data sources and protein functions. In this study, we use ten different genomic data sources in <it>Mus musculus</it>, including: protein-domains, protein-protein interactions, gene expressions, phenotype ontology, phylogenetic profiles and disease data sources to predict protein functions that are labelled with Gene Ontology (GO) terms. We then apply two approaches to feature selection: exhaustive search feature selection using a kernel based logistic regression (KLR), and a kernel based <it>L1</it>-norm regularized logistic regression (KL1LR). In the first approach, we exhaustively measure the contribution of each data set for each function based on its prediction quality. In the second approach, we use the estimated coefficients of features as measures of contribution of data sources. Our results show that the proposed methods improve the prediction quality compared to the full integration of all data sources and other filter-based feature selection methods. We also show that contributing data sources can differ depending on the protein function. Furthermore, we observe that highly contributing data sets can be similar among a group of protein functions that have the same parent in the GO hierarchy.</p> <p>Conclusions</p> <p>In contrast to previous integration methods, our approaches not only increase the prediction quality but also gather information about highly contributing data sources for each protein function. This information can help researchers collect relevant data sources for annotating protein functions.</p

WNT1-induced Secreted Protein-1 (WISP1), a Novel Regulator of Bone Turnover and Wnt Signaling

WISP1/CCN4 (hereafter referred to as WISP1), a member of the CCN family, is found in mineralized tissues and is produced by osteoblasts and their precursors. In this study, Wisp1-deficient (Wisp1(−/−)) mice were generated. Using dual-energy x-ray absorptiometry, we showed that by 3 months, the total bone mineral density of Wisp1(−/−) mice was significantly lower than that of WT mice. Further investigation by micro-computed tomography showed that female Wisp1(−/−) mice had decreased trabecular bone volume/total volume and that both male and female Wisp1(−/−) mice had decreased cortical bone thickness accompanied by diminished biomechanical strength. The molecular basis for decreased bone mass in Wisp1(−/−) mice arises from reduced bone formation likely caused by osteogenic progenitors that differentiate poorly compared with WT cells. Osteoclast precursors from Wisp1(−/−) mice developed more tartrate-resistant acid phosphatase-positive cells in vitro and in transplants, suggesting that WISP1 is also a negative regulator of osteoclast differentiation. When bone turnover (formation and resorption) was induced by ovariectomy, Wisp1(−/−) mice had lower bone mineral density compared WT mice, confirming the potential for multiple roles for WISP1 in controlling bone homeostasis. Wisp1(−/−) bone marrow stromal cells had reduced expression of β-catenin and its target genes, potentially caused by WISP1 inhibition of SOST binding to LRP6. Taken together, our data suggest that the decreased bone mass found in Wisp1(−/−) mice could potentially be caused by an insufficiency in the osteodifferentiation capacity of bone marrow stromal cells arising from diminished Wnt signaling, ultimately leading to altered bone turnover and weaker biomechanically compromised bones