The role of CCN2 in cartilage and bone development

Abstract

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.

Fig. 1

Molecular action of CCN2 in cartilage. CCN2 molecule represented by the complex with 4 spheres in the center interacts with a number of biomolecules in different categories, which results in the integrated manipulation of extracellular signaling. The letters “I,” “V,” “T,” and “C” on the spheres denote insulin-like growth factor-like module, von Willebrand factor type C repeat, thrombospondin type 1 repeat and C-terminal cystine knot module, respectively. Growth factors interacting with CCN2 include transforming growth factor beta (TGF-β), bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF). Binding between CCN2 and cartilaginous ECM molecules, such as fibronectin (Fn) and aggrecan, is indicated. Several cell-surface receptors present on chondrocytes as represented by integrins (Itg), low-density lipoprotein receptor related protein 1 (LRP-1) and Trk A are known to directly interact with CCN2. Of note, recent investigation is revealing the interaction of CCN2 with another CCN family member, as well as with itself

Fig. 2

Classification of cartilage present in vertebrates. Temporary cartilage appears during development to accomplish skeletal formation and growth. A class of temporary cartilage, here termed bone-directed cartilage, itself is mineralized to become bone under the collaboration with osteoblasts and vascular endothelial cells. In the case of bone-inducing cartilage, bone is formed independently at a space in close proximity. Permanent cartilage is formed and maintained to add elasticity and stiffness required in certain organs. Based on the physical property, histological characteristics, and ECM composition, permanent cartilage can be classified as hyaline, elastic or fibrous

Fig. 3

Endochondral ossification by growth plate cartilage and role of CCN2. Inside of long bones, chondrocytes in the growth plate undergo a series of differentiation processes to execute the bone growth. CCN2 is mainly produced by growth plate chondrocytes in the prehypertrophic and hypertrophic layers and infiltrates through the ECM in both directions. Chondrocytes located most distantly from the CCN2 producers are less stimulated by CCN2 and stay at rest. Those located nearby the prehypertrophic ones are strongly stimulated in a matricrine manner to proliferate and maturate to produce cartilaginous ECM, which confers longitudinal bone growth. Prehypertrophic chondrocytes themselves are stimulated by CCN2 in an autocrine/intracrine manner and forwarded to terminal differentiation toward apoptosis. CCN2 released to the bone marrow acts on osteoblasts, vascular endothelial (VE) cells, and osteoclasts to promote the replacement of cartilaginous tissue with vascularized bone and bone remodeling. C: CCN2 molecule

Fig. 4

CCN2 in articular cartilage under physiological and pathological situations. CCN2 is known to promote all of the physiological steps towards articular cartilage formation without promoting pathological hypertrophic changes in articular chondrocytes (right). In osteoarthritis (OA) cartilage, CCN2 production is strongly induced in articular chondrocytes in order to increase the cell number and compensate the ECM deficiency, which results in the formation of the chondrocyte clusters typically observed in OA cartilage. This hypothetical action of CCN2 in articular cartilage is strongly supported by the fact that CCN2 regenerates the tissue lost in a full-thickness defect of articular cartilage in vivo. C: CCN2 molecule

Fig. 5

Transcriptional and post-transcriptional regulation of the CCN2 gene in chondrocytes. Transcriptional regulation observed in chondrocytes or chondrocytic cells is summarized in the upper-half, while post-transcriptional regulation is schematized in the lower half. TGF-β is known to enhance the CCN2 expression in chondrocytes, as well as in a number of other types of cells. This regulation is supposedly mediated by 3 genetic cis-elements: Smad-binding element (SBE), basal control element (BCE), and Ets1-binding sequence in the proximal promoter, directly or indirectly through endothelin-1 (ET-1). Hatched arrows indicate the actions experimentally indicated from studies using non-chondrocytic cells. MMP-3 is shown to directly bind to TRENDIC to enhance transcription. The RNA stability and translation of the CCN2 mRNA is regulated by direct interaction with nucleophosmin (NPM) and miR-18a through their specific targets in the 3′-untranslated region (UTR). The cis-acting element of structure-anchored repression (CAESAR) mediates the post-transcriptional regulation by factor(s) yet to be identified. TATA, ORF and AAAAA represent the TATA box, open reading frame and polyadenyl tail, respectively