To understand the molecular events coupling between cell proliferation and differentiation by elucidating genes essential for the process, we conducted a large scale gene expression analysis of an in vitro osteoclastogenesis system consisting of recombinant RANKL and mouse RAW264 cells. The entire process leading to the formation of tartrate resistant acid phosphatase-positive multinucleated cells takes 3 days and plates become fully covered with multinucleated cells at 4 days. Microarray probing at eight time points revealed 635 genes that showed greater than 2-fold differential expression for at least one time point and they could be classified into six groups by the "k-means" clustering analysis. Among a group of 106 early inducible genes (within 2-5 h after RANKL stimulation), four genes including NFAT2 were identified as genes whose enhanced expressions were fairly correlated with an efficient induction of matured osteoclasts. Moreover, cyclosporin A significantly suppressed the multinucleated cell formation accompanying the reduction of the nuclear localization of NFAT2. When the expression of NFAT2 was suppressed by introducing antisense NFAT2, multinucleated cell formation was severely hampered. Functional analysis thus combined with gene analysis by microarray technology elucidated a key role of NFAT2 in osteoclastogenesis in vitro.Specific factors/regulatory genes playing essential roles for cellular differentiation have been identified in various systems, and they have been shown to exert their effects eventually through the induction or repression of certain groups of genes (1-4). Therefore, gene expression profiling based on fine statistical analysis in addition to an elucidation of key factors/ genes might be essential to understand the molecular mechanisms underlying the differentiation process of a certain cell type. Fortunately, recent advances in the technology for assaying RNA in a highly parallel fashion (5-7), coupled with the completion/progress of several mammalian genome projects, make the approach feasible if a refined system is available. Here, we describe the broad outlines of gene expression during osteoclastogenesis in vitro, in particular during the initial stage, and explain the identification and characterization of genes essential for osteoclastogenesis on the basis of profiling characteristics. A similar approach using a different cell system was reported recently (8).Osteoclasts are multinucleated (MN) 1 giant cells and present only in bone with the capacity to resorb mineralized tissues (9). They were reported to be formed by fusion of mononuclear precursor cells derived from colony-forming unit granulocyte macrophages (CFU-GM) and branch from the monocyte-macrophage lineage during the early stage of the differentiation process (9, 10). Recently, a key factor responsible for initiating this differentiation process was identified and named receptor activator of NFB ligand (RANKL) (or osteoclast differentiation factor (ODF)/TNF-related activation-induced cytokine TRANCE) (11-13)...

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-twohybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcomaderived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/ CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGFenhancing effect on cell adhesion to FN was abolished by a blocking antibody against a5b1 integrin (a5b1), but not by one against anti-avb3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that a5b1 is involved in this adhesion.

CCN2/CTGF (CCN family 2/connective tissue growth factor) is a multi-cellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production, and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF-interactive proteins capable of modulating its action in cartilage, we carried out a yeast two-hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. In the present paper, we report the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, as a CCN2/CTGF-binding protein. Among the four domains of CCN2/CTGF, the IGFBP [IGF (insulin-like growth factor)-binding protein-like] and/or VWC (von Willebrand factor type C) domains had a direct interaction with aggrecan in a yeast two-hybrid assay. The results of a solid-phase-binding assay using aggrecan-coated plates also showed binding to recombinant CCN2/CTGF in a dose-dependent manner. rIGFBP (recombinant IGFBP) and rVWC (recombinant VWC) module peptides had stronger binding to aggrecan compared with rTSP1 (recombinant thrombospondin type 1 repeat) and rCT (recombinant C-terminal cystine knot) module peptides. SPR (surface plasmon resonance) analysis showed the direct interaction between the CCN2/CTGF and aggrecan, and ectopically overexpressed CCN2/CTGF and AgG3 (G3 domain of aggrecan) confirmed their binding In vivo. Indirect immunofluorescence analysis indicated that CCN2/CTGF was extracellularly co-localized with aggrecan on HCS-2/8 cells. The rIGFBP–rVWC peptide effectively enhanced the production and release of aggrecan compared with the rTSP–rCT peptide in chondrocytes. These results indicate that CCN2/CTGF binds to aggrecan through its N-terminal IGFBP and VWC modules, and this binding may be related to the CCN2/CTGF-enhanced production and secretion of aggrecan by chondrocytes.

Sclerostin (Scl) negatively regulates bone formation and favors bone resorption. Osteocytes, the cells responsible for mechanosensing, are known as the primary source of Scl and are a key regulator of bone remodeling through the induction of receptor activator of NF-κB ligand (RANKL). However, the spatiotemporal patterns of Scl in response to mechanical stimuli and their regulatory mechanisms remain unknown. We investigated the regulatory dynamics of the SOST/Scl expression generated by orthodontic tooth movement (OTM) in vivo and in vitro. In 8-wk-old male mice, coil springs were used to move the first molar mesially for 0, 1, 5, or 10 d. A regional histogram and the distribution patterns of the Scl expression showed that the Scl expression in the alveolar bone was increased on the compression side and peaked on day 5, with a gradual increase in the degree of significance. On day 10, the expression around the periodontal ligament (PDL)-alveolar bone boundary returned to the control level. Conversely, the expression of Scl on the tension side was only significantly decreased on day 1. Compressive force biphasically modulated the SOST/Scl expression in the isolated human PDL and thereby upregulated osteocytic SOST via paracrine activation in an osteocyte-PDL co-culture system designed to mimic OTM. This system did not affect the RANKL or OPG expression in osteocytes, suggesting that the bone resorption pathways are acted upon in a PDL-dependent and osteocyte-independent manner through RANKL/OPG signaling. Moreover, sclerostin neutralizing antibody significantly attenuated the upregulation of SOST that was induced by compressive force. In conclusion, our results provide evidence to support that factors secreted by the PDL, including SOST/Scl, control alveolar bone remodeling through osteocytic SOST/Scl in OTM.

To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2 ⁄ 8. CCN2 ⁄ CTGF and CCN3 ⁄ NOV polypeptides were picked up as CCN2-binding proteins, and CCN2-CCN2 and CCN2-CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (K d ) were 1.17 · 10 )9 M for CCN2 and CCN2, and 1.95 · 10 )9 M for CCN2 and CCN3. Ectopically overexpressed green fluorescent protein-CCN2 and Halo-CCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2-CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2 ⁄ 8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3.