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/CTGF is a multifunctional factor that plays a crucial role in the growth and differentiation of chondrocytes. The chicken ccn2 gene is regulated not only at the transcriptional level but also by the interaction between a posttranscriptional element in the 3 untranslated region (3-UTR) and a cofactor. In the present study, we identified a nucleophosmin (NPM) (also called B23) as this cofactor. Binding of NPM to the element was confirmed, and subsequent analysis revealed a significant correlation between the decrease in cytosolic NPM and the increased stability of the ccn2 mRNA during chondrocyte differentiation in vivo. Furthermore, recombinant chicken NPM enhanced the degradation of chimeric RNAs containing the posttranscriptional cis elements in a chicken embryonic fibroblast extract in vitro. It is noteworthy that the RNA destabilization effect by NPM was far more prominent in the cytosolic extract of chondrocytes than in that of fibroblasts, representing a chondrocyte-specific action of NPM. Stimulation by growth factors to promote differentiation changed the subcellular distribution of NPM in chondrocytes, which followed the expected patterns from the resultant change in the ccn2 mRNA stability. Therefore, the present study reveals a novel aspect of NPM as a key player in the posttranscriptional regulation of ccn2 mRNA during the differentiation of chondrocytes. CCN2 (connective tissue growth factor [CTGF]/hypertrophic chondrocyte-specific gene product 24 [Hcs24]) is a cysteine-rich secretory protein of 36 to 38 kDa that has four distinct modules, i.e., insulin-like growth factor-binding protein-likes, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and C-terminal modules (4,6,54,68,69). CCN2 is a member of the CCN family (reviewed in references 6, 38, 53, 54, 68, and 69), which comprises ccn1 (cef-10/cyr61 [37,66]), ccn2 (ctgf/hcs24/fisp12 [5,46,57]), ccn3 (nov [25]), ccn4 (elm-1/wisp-1 [20,52]), ccn5 (ctgf-l/wisp-2/cop1 [52,75]), and ccn6 (wisp-3 [52]). CCN2 was initially isolated from angioendothelial cells as a growth factor related to plateletderived growth factor (PDGF) and was revealed to have PDGF-like mitogenic and chemotactic activities toward fibroblasts (17,23,28,33,54). However, recent studies have revealed that CCN2 is a multifunctional factor that regulates the growth and/or differentiation, chemotaxis, adhesion of various cells, and extracellular matrix formation by various cells, including vascular endothelial cells (2,29,54,64). Furthermore, we showed earlier that CCN2 plays an important role in the growth and differentiation of chondrocytes and osteoblasts during endochondral ossification (46-48).As to CCN2, it has been suggested that its gene expression is regulated at multiple steps, such as transcriptional, posttranscriptional, and translational stages (1), for playing its multiple roles mentioned above. For instance, transforming growth factor ␤ (TGF-␤) induces the expression of CCN2 (28,46), and a few TGF-␤ response elements have been found in the ccn2 promote...

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Periodontal tissue homeostasis depends on a complex cellular network that conveys cell-cell communication. Gap junctions (GJs), one of the intercellular communication systems, are found between adjacent human periodontal ligament (hPDL) cells; however, the functional GJ coupling between hPDL cells has not yet been elucidated. In this study, we investigated functional gap-junction-mediated intercellular communication in isolated primary hPDL cells. SEM images indicated that the cells were in contact with each other via dendritic processes, and also showed high anti-connexin43 (Cx43) immunoreactivity on these processes. Gap-junctional intercellular communication (GJIC) among hPDL cells was assessed by fluorescence recovery after a photobleaching (FRAP) analysis, which exhibited dye coupling between hPDL cells, and was remarkably down-regulated when the cells were treated with a GJ blocker. Additionally, we examined GJs under hypoxic stress. The fluorescence recovery and expression levels of Cx43 decreased time-dependently under the hypoxic condition. Exposure to GJ inhibitor or hypoxia increased RANKL expression, and decreased OPG expression. This study shows that GJIC is responsible for hPDL cells and that its activity is reduced under hypoxia. This is consistent with the possible role of hPDL cells in regulating the biochemical reactions in response to changes in the hypoxic environment.

EphA4 receptor tyrosine kinase has been shown to be critically involved in neural tissue development. Here, we found EphA4 was also distributed among hypertrophic chondrocytes and osteoblasts in the growth plate of developing mouse long bones. In vitro evaluation revealed that ephA4 expression was elevated upon hypertrophic differentiation of chondrocytes and that markedly stronger expression was observed in osteoblastic SaOS-2 than chondrocytic HCS-2/8 cells. Of note, RNAi-mediated silencing of ephA4 in SaOS-2 cells resulted in the repression of osteocalcin gene expression and alkaline phosphatase activity. Interestingly, confocal laser-scanning microscopic analysis revealed the presence of EphA4 molecules in the nucleus as well as on the surface of SaOS-2 cells. These findings are the first indication of a critical role of EphA4 in ossification, especially at the final stage in which osteoblasts and hypertrophic chondrocytes play major roles.