軟骨細胞におけるLRP1，CCN2受容体の機能の可能性

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

Molecular and Genetic Interactions between CCN2 and CCN3 behind Their Yin-Yang Collaboration

Cellular communication network factor (CCN) 2 and 3 are the members of the CCN family that conduct the harmonized development of a variety of tissues and organs under interaction with multiple biomolecules in the microenvironment. Despite their striking structural similarities, these two members show contrastive molecular functions as well as temporospatial emergence in living tissues. Typically, CCN2 promotes cell growth, whereas CCN3 restrains it. Where CCN2 is produced, CCN3 disappears. Nevertheless, these two proteins collaborate together to execute their mission in a yin-yang fashion. The apparent functional counteractions of CCN2 and CCN3 can be ascribed to their direct molecular interaction and interference over the cofactors that are shared by the two. Recent studies have revealed the mutual negative regulation systems between CCN2 and CCN3. Moreover, the simultaneous and bidirectional regulatory system of CCN2 and CCN3 is also being clarified. It is of particular note that these regulations were found to be closely associated with glycolysis, a fundamental procedure of energy metabolism. Here, the molecular interplay and metabolic gene regulation that enable the yin-yang collaboration of CCN2 and CCN3 typically found in cartilage development/regeneration and fibrosis are described

Retrotransposons Manipulating Mammalian Skeletal Development in Chondrocytes

Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed

Expression and function of CCN2-derived circRNAs in chondrocytes

Cellular communication network factor 2 (CCN2) molecules promote endochondral ossification and articular cartilage regeneration, and circular RNAs (circRNAs), which arise from various genes and regulate gene expression by adsorbing miRNAs, are known to be synthesized from CCN2 in human vascular endothelial cells and other types of cells. However, in chondrocytes, not only the function but also the presence of CCN2-derived circRNA remains completely unknown. In the present study, we investigated the expression and function of CCN2-derived circRNAs in chondrocytes. Amplicons smaller than those from known CCN2-derived circRNAs were observed using RT-PCR analysis that could specifically amplify CCN2-derived circRNAs in human chondrocytic HCS-2/8 cells. The nucleotide sequences of the PCR products indicated novel circRNAs in the HCS-2/8 cells that were different from known CCN2-derived circRNAs. Moreover, the expression of several Ccn2-derived circRNAs in murine chondroblastic ATDC5 cells was confirmed and observed to change alongside chondrocytic differentiation. Next, one of these circRNAs was knocked down in HCS-2/8 cells to investigate the function of the human CCN2-derived circRNA. As a result, CCN2-derived circRNA knockdown significantly reduced the expression of aggrecan mRNA and proteoglycan synthesis. Our data suggest that CCN2-derived circRNAs are expressed in chondrocytes and play a role in chondrogenic differentiation

Regulation of cellular communication network factor 2 (CCN2) in breast cancer cells via the cell-type dependent interplay between CCN2 and glycolysis

Objectives: Anti-osteoclastic treatments for breast cancer occasionally cause medication-related osteonecrosis of the jaw. Moreover, elevated glycolytic activity, which is known as the Warburg effect, is usually observed in these breast cancer cells. Previously, we found that cellular communication network factor 2 (CCN2) production and glycolysis enhanced each other in chondrocytes. Here, we evaluated the interplay between CCN2 and glycolysis in breast cancer cells, as we suspected a possible involvement of CCN2 in the Warburg effect in highly invasive breast cancer cells. Methods: Two human breast cancer cell lines with a distinct phenotype were used. Glycolysis was inhibited by using 2 distinct compounds, and gene silencing was performed using siRNA. Glycolysis and the expression of relevant genes were monitored via colorimetric assays and quantitative RT-PCR, respectively. Results: Although CCN2 expression was almost completely silenced when treating invasive breast cancer cells with a siRNA cocktail against CCN2, glycolytic activity was not affected. Notably, the expression of glycolytic enzyme genes, which was repressed by CCN2 deficiency in chondrocytes, tended to increase upon CCN2 silencing in breast cancer cells. Inhibition of glycolysis, which resulted in the repression of CCN2 expression in chondrocytic cells, did not alter or strongly enhanced CCN2 expression in the invasive and non-invasive breast cancer cells, respectively. Conclusions: High CCN2 expression levels play a critical role in the invasion and metastasis of breast cancer. Thus, a collapse in the intrinsic repressive machinery of CCN2 due to glycolysis may induce the acquisition of an invasive phenotype in breast cancer cells

Odontoblast differentiation is regulated by an interplay between primary cilia and the canonical Wnt pathway

Primary cilium is a protruding cellular organelle that has various physiological functions, especially in sensory reception. While an avalanche of reports on primary cilia have been published, the function of primary cilia in dental cells remains to be investigated. In this study, we focused on the function of primary cilia in dentin-producing odontoblasts. Odontoblasts, like most other cell types, possess primary cilia, which disappear upon the knockdown of intraflagellar transport-88. In cilia-depleted cells, the expression of dentin sialoprotein, an odontoblastic marker, was elevated, while the deposition of minerals was slowed. This was recapitulated by the activation of canonical Wnt pathway, also decreased the ratio of ciliated cells. In dental pulp cells, as they differentiated into odontoblasts, the ratio of ciliated cells was increased, whereas the canonical Wnt signaling activity was repressed. Our results collectively underscore the roles of primary cilia in regulating odontoblastic differentiation through canonical Wnt signaling. This study implies the existence of a feedback loop between primary cilia and the canonical Wnt pathway

Promotion of Bone Regeneration by CCN2 Incorporated into Gelatin Hydrogel

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

Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells

Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates alpha 1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells