Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of matricellular proteins and also has been designated Hcs24, FISP12, IGFBP8, IGFBP-rP2, ␤IG-M2, and ecogenin. The other CCN proteins include Cyr61/CCN1, NOV/CCN3, WISP1/CCN4, WISP2/CCN5, and WISP3/CCN6 (5, 26, 38, 39) as well, and they are structurally and functionally related glycoproteins involved in cell differentiation, proliferation, adhesion, migration, and the formation of the extracellular matrix. These matricellular functions of CCNs are involved in physiological processes such as wound healing, angiogenesis, morphogenesis, and embryogenesis as well as in pathological states including fibrotic disorders, cancer, and arthritis.Earlier we showed that CCN2 promotes endochondral ossification by acting on chondrocytes, osteoblasts, and endothelial cells (35,37,46). For example, CCN2 promotes physiological chondrocytic proliferation and extracellular matrix (ECM) formation. We also reported the regeneration of defects in articular cartilage in rat knee joints following treatment with recombinant CCN2 (36). Furthermore, ctgf-null mice were dead on delivery and were characterized by defective angiogenesis, the derangement of endochondral ossification, and dysmorphisms that occurred as a result of impaired chondrocyte proliferation and an abnormal ECM composition within the hypertrophic zone (24).Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are involved in the remodeling and turnover of the ECM in physiological processes such as angiogenesis, wound healing, embryogenesis, and morphogenesis as well as in pathological states including cancers, myocardial infarction, fibrotic disorders, rheumatism, and osteoarthritis (33, 49). Cartilage is a connective tissue that is constructed by chondrocytes embedded within an ECM predominantly composed of collagens and proteoglycans. ECM remodeling is achieved by regulating the production and degradation of specific ECM components. MMPs, which comprise a large family of enzymes with differential abilities to degrade specific ECM components, play a vital role in this process. MMPs also cleave growth factors and their binding proteins, thereby activating or inhibiting specific signaling events (15). Of note, the expression and role of MMP3 have been investigated in the pathological status of articular cartilage, such as in osteoarthritis and rheumatism (1,52).Recent study has demonstrated the existence and functions of intracellular MMPs and tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 accumulates in the cellular nuclei in association with the cell cycle (54). Alternative splicing and promoter usage generate an intracellular MMP11 isoform directly translated as an active MMP (31). MMP2 is found in the nuclei of cardiac myocytes and is capable of cleaving poly-(ADP-ribose) polymerase (PARP) in vitro (28). MMP3 also is detected in the nuclei of hepatocytes and is involved in apop-* Corresponding author. Mailing address:

Wound healing and tissue regeneration are usually initiated by coagulation followed by fibrous tissue formation. In the present study, we discovered an abundance of connective tissue growth factor (CTGF/CCN2) in human platelets, which was released along with the coagulation process. The CTGF/CCN2 content in platelets was 10-fold higher than that in arterial tissue. Furthermore, the CTGF/CCN2 content in a single platelet was computed to be more than 20-fold higher than that of any other growth factor reported. Considering that CTGF/CCN2 promotes angiogenesis, cartilage regeneration, fibrosis and platelet adhesion, it may be now regarded as one of the major functional components of platelets.

CCN4/Wnt‐induced secreted protein 1 (WISP1) is one of the CCN (CTGF/Cyr61/Nov) family proteins. CCN members have typical structures composed of four conserved cysteine‐rich modules and their variants lacking certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4/WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. The expression of CCN4/WISP1 mRNA and its variants were analyzed in a human chondrosarcoma‐derived chondrocytic cell line, HCS‐2/8, and primary rabbit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS‐2/8, as well as full‐length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4/WISP1 mRNA along chondrocyte differentiation, RGC cells were cultured and sampled until they were mineralized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal differentiation in HCS‐2/8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS‐2/8‐specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas.

Low density lipoprotein receptor (LDLR)-related protein 1 (LRP1/CD91) is one of the receptors of CCN2 that conducts endochondral ossification and cartilage repair. LRP1 is a well-known endocytic receptor, but its distribution among chondrocytes remains to be elucidated. We herein demonstrate for the first time that the distribution of LRP1 in chondrocytes except for hypertrophic chondrocytes in vivo and in vitro. Interestingly, the LRP1 levels were higher in mature chondrocytic HCS-2/8 and osteoblastic SaOS-2 than in other cells, whereas the other LDLR family members involved in ossification were detected at lower levels in HCS-2/8. It was interesting to note that in HCS-2/8, LRP1 was observed not only on the cell surface and in the cytoplasm, but also in the nucleus. Exogenously added CCN2 was incorporated into HCS-2/8, which was partially co-localized with LRP1, and targeted to the recycling endosomes and nucleus as well as the lysosomes. These findings suggest specific roles of LRP1 in cartilage biology.

Micro RNA (miRNA) is a small non-coding post-transcriptional RNA regulator that is involved in a variety of biological events. In order to specify the role of miRNAs in cartilage metabolism, we comparatively analyzed the expression profile of known miRNAs in chicken sternum chondrocytes representing early and late differentiation stages. Interestingly, none of the miRNAs displaying strong expression levels showed remarkable changes along with differentiation, suggesting their roles in maintaining the homeostasis rather than cytodifferentiation of chondrocytes. Among these miRNAs, miR-181a, which is known to play critical roles in a number of tissues, was selected and was further characterized. Human microarray analysis revealed remarkably stronger expression of miR-181a in human HCS-2/8 cells, which strongly maintained a chondrocytic phenotype, than in HeLa cells, indicating its significant role in chondrocytes. Indeed, subsequent investigation indicated that miR-181a repressed the expression of two genes involved in cartilage development. One was CCN family member 1 (CCN1), which promotes chondrogenesis; and the other, the gene encoding the core protein of aggrecan, a major cartilaginous proteoglycan, aggrecan. Based on these findings, negative feedback system via miR-181a to conserve the integrity of the cartilaginous phenotype may be proposed.

CCN2/connective tissue growth factor (CTGF) is a unique molecule that promotes both chondrocytic differentiation and proliferation through its matricellular interaction with a number of extracellular biomolecules. This apparently contradictory functional property of CCN2 suggests its certain role in basic cellular activities such as energy metabolism, which is required for both proliferation and differentiation. Comparative metabolomic analysis of costal chondrocytes isolated from wild-type and Ccn2-null mice revealed overall impaired metabolism in the latter. Among the numerous metabolites analyzed, stable reduction in the intracellular level of ATP, GTP, CTP, or UTP was observed, indicating a profound role of CCN2 in energy metabolism. Particularly, the cellular level of ATP was decreased by more than 50% in the Ccn2-null chondrocytes. The addition of recombinant CCN2 (rCCN2) to cultured Ccn2-null chondrocytes partly redeemed the cellular ATP level attenuated by Ccn2 deletion. Next, in order to investigate the mechanistic background that mediates the reduction in ATP level in these Ccn2-null chondrocytes, we performed transcriptome analysis. As a result, several metabolism-associated genes were found to have been up-regulated or down-regulated in the mutant mice. Up-regulation of a number of ribosomal protein genes was observed upon Ccn2 deletion, whereas a fewgenes required for aerobic and anaerobic ATP production were down-regulated in the Ccn2-null chondrocytes. Among such genes, reduction in the expression of the enolase 1 gene was of particular note. These findings uncover a novel functional role of CCN2 as a metabolic supporter in the growth-plate chondrocytes, which is required for skeletogenesis in mammals.

SummaryLow-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-b-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a unique molecule that promotes the entire endochondral ossification process and regeneration of damaged articular cartilage. Also, CCN2 has been shown to enhance the adhesion and migration of bone marrow stromal cells as well as the growth and differentiation of osteoblasts; hence, its utility in bone regeneration has been suggested. Here, we evaluated the effect of CCN2 on the regeneration of an intractable bone defect in a rat model. First, we prepared two recombinant CCN2s of different origins, and the one showing the stronger effect on osteoblasts in vitro was selected for further evaluation, based on the result of an in vitro bioassay. Next, to obtain a sustained effect, the recombinant CCN2 was incorporated into gelatin hydrogel that enabled the gradual release of the factor. Evaluation in vivo indicated that CCN2 continued to be released at least for up to 14 days after its incorporation. Application of the gelatin hydrogel-CCN2 complex, together with a collagen scaffold to the bone defect prepared in a rat femur resulted in remarkable induction of osteoblastic mineralization markers within 2 weeks. Finally, distinct enhancement of bone regeneration was observed 3 weeks after the application of the complex. These results confirm the utility of CCN2 in the regeneration of intractable bone defects in vivo when the factor is incorporated into gelatin hydrogel.