Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

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. Production and role of CCN2-derived RNAs in chondrocytes.

Dual roles of cellular communication network factor 6 (CCN6) in the invasion and metastasis of oral cancer cells to bone via binding to BMP2 and RANKL.

The acquisition of motility via epithelial-mesenchymal transition (EMT) and osteoclast induction are essential for the invasion and metastasis of oral squamous cell carcinoma (OSCC) to bone. However, the molecule suppressing both EMT and osteoclastogenesis is still unknown. In this study, we found that cellular communication network factor 6 (CCN6) was less produced in a human OSCC cell line, HSC-3 with mesenchymal phenotype, than in HSC-2 cells without it. Notably, CCN6 interacted with bone morphogenetic protein 2 (BMP2) and suppressed the cell migration of HSC-3 cells stimulated by BMP2. Moreover, knockdown of CCN6 in HSC-2 cells led to the promotion of EMT and enhanced the effect of transforming growth factor-ß (TGF-ß) on the promotion of EMT. Furthermore, CCN6 combined with BMP2 suppressed EMT. These results suggest that CCN6 strongly suppresses EMT in cooperation with BMP2 and TGF-ß. Interestingly, CCN6 combined with BMP2 increased the gene expression of receptor activator of nuclear factor-κB ligand (RANKL) in HSC-2 and HSC-3 cells. Additionally, CCN6 interacted with RANKL, and CCN6 combined with RANKL suppressed RANKL-induced osteoclast formation. In metastatic lesions, increasing BMP2 due to the bone destruction led to interference with binding of CCN6 to RANKL, which results in the promotion of bone metastasis of OSCC cells due to continuous osteoclastogenesis. These findings suggest that CCN6 plays dual roles in the suppression of EMT and in the promotion of bone destruction of OSCC in primary and metastatic lesions, respectively, through cooperation with BMP2 and interference with RANKL.

Do not overwork: cellular communication network factor 3 for life in cartilage.

Cellular communication network factor (CCN) 3, which is one of the founding members of the CCN family, displays diverse functions. However, this protein generally represses the proliferation of a variety of cells. Along with skeletal development, CCN3 is produced in cartilaginous anlagen, growth plate cartilage and epiphysial cartilage. Interestingly, CCN3 is drastically induced in the growth plates of mice lacking CCN2, which promotes endochondral ossification. Notably, chondrocytes in these mutant mice with elevated CCN3 production also suffer from impaired glycolysis and energy metabolism, suggesting a critical role of CCN3 in cartilage metabolism. Recently, CCN3 was found to be strongly induced by impaired glycolysis, and in our study, we located an enhancer that mediated CCN3 regulation via starvation. Subsequent investigations specified regulatory factor binding to the X-box 1 (RFX1) as a transcription factor mediating this CCN3 regulation. Impaired glycolysis is a serious problem, resulting in an energy shortage in cartilage without vasculature. CCN3 produced under such starved conditions restricts energy consumption by repressing cell proliferation, leading chondrocytes to quiescence and survival. This CCN3 regulatory system is indicated to play an important role in articular cartilage maintenance, as well as in skeletal development. Furthermore, CCN3 continues to regulate cartilage metabolism even during the aging process, probably utilizing this regulatory system. Altogether, CCN3 seems to prevent "overwork" by chondrocytes to ensure their sustainable life in cartilage by sensing energy metabolism. Similar roles are suspected to exist in relation to systemic metabolism, since CCN3 is found in the bloodstream.

CCN Proteins (Cellular Communication Network Factors): Expanding Their Repertoire Toward a New Concept.

I herein report the general structures and functions of CCN proteins and possible molecular mechanisms involved in the unique biological actions of this family of intercellular signaling regulators, which are considered matricellular proteins and were once referred to as "signal conductors" but have recently been renamed "Cellular Communication Network Factors." Their repertoire of functions beyond their role as matricellular proteins is also described to aid in future studies. Advanced research concerning their relevance to pathology is briefly introduced as well. The information provided in this chapter is expected to be useful for readers of subsequent chapters.

Cellular Fluorescence Imaging for the Evaluation of Bioactivity of CCN Family Proteins.

The method of labeling proteins of interest with fluorescent dyes that can specifically stain organelles in living cells provides a tool for investigating various cellular processes under a microscope. Visualization (imaging) of the cells using fluorescence has many advantages, including the ability to stain multiple cell organelles and intracellular proteins simultaneously and discriminately, and is used in many research fields. In this chapter, we describe the observation of cell organelles using fluorescence staining to analyze the functions of CCN family proteins involved in various cellular events.

Imaging of Molecular Interaction Between CCN Protein and Its Binding Partners: An In Situ Proximity Ligation Assay of Interaction Between CCN2 and Rab14 in Chondrocytes.

An in situ proximity ligation assay (PLA) enables visualization of protein interactions in fixed cells. It is a powerful method for investigating protein-protein binding of endogenously expressed proteins. To confirm binding between CCN2 and Rab14 GTPase (Rab14) in chondrocytes, we performed a PLA using chondrocytic HCS-2/8 cells. The protocol in this chapter introduces an optimized technique for visualizing intracellular interactions of CCN2 and Rab14 in fixed cells using a PLA.

Evaluation of the Molecular Interaction Between CCN Protein and Its Binding Partners: A Solid-Phase Binding Assay and Surface Plasmon Resonance.

CCN proteins are known to bind to various growth factors, cytokines, and membrane proteins. Since these bindings are closely involved in the function of CCN proteins, the analysis of the binding partners is the first step toward understanding the mechanisms of actions of CCN proteins. This chapter describes two approaches used for such analyses: a solid-phase binding assay, which is suitable for confirming the binding easily because of its simplicity and cost advantage, and a surface plasmon resonance assay, which can determine the binding affinities between CCN proteins and their partners.

Protocols for Screening Peptides Binding to CCN Family Proteins and Their Extended Utility.

The function of CCN family proteins is determined by their interactions with multiple cofactors that are present in the microenvironment. Therefore, determining these cofactors is critically important in understanding the molecular function of CCN family members. For this objective, a bacteriophage random peptide display library is a suitable tool. In this library, each filamentous bacteriophage is designed to display an oligopeptide of 7-20 random amino acid residues on its surface. Bacteriophage clones that possess peptides that bind to a CCN family protein are selected through several cycles of a process called biopanning or affinity selection. By determining the nucleotide sequence of the DNA that encodes the displayed peptide, the oligopeptides that specifically bind to the CCN family member can be specified. The obtained peptide sequences can be utilized to design peptide aptamers for CCN family proteins, or as a key sequence to determine new CCN family cofactor candidates in silico. Instead of a random peptide cDNA library, an antibody cDNA library from naïve lymphocytes or from B cells immunized by a CCN family protein can be used in order to obtain a highly specific CCN family detection or functional modulation tool.

Analyses of the Posttranscriptional Regulation of CCN Genes: Approach to Multiple Steps of CCN2 Gene Expression.

Cells generally control the concentration of mRNA via transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation (decay) cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, degradation of the target mRNA, and inhibition of mRNA translation are performed either individually or in combination. From our experience, measurement of the steady-state levels of mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying the ccn2 gene expression. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. The stability of ccn2 mRNAs is then evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Finally, repression of ccn2 mRNA translation can be estimated by comparing the expression of mRNA and protein changes. We herein report the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can, in theory, be used to elucidate the posttranscriptional regulation of other genes belonging to the CCN family.

The Evaluation of Meniscus Regenerative Effects of LIPUS-Induced CCN Proteins: Induction by LIPUS of CCN2 and Meniscus-Related Genes in Cultured Meniscus Cells and Meniscus Tissues.

Menisci are a pair of crescent-shaped fibrocartilages and composed primarily of type I collagen. Inner region of the meniscus has similar characteristics to articular cartilage. Low-intensity pulsed ultrasound (LIPUS) has been reported to have chondroprotective effects on chondrocytes by inducing the expression of chondrocyte differentiation markers and CCN2/CTGF production. Here, we describe an experimental approach that investigates the distinct cellular behavior of human inner and outer meniscus cells in response to LIPUS stimulation. Our experimental model can analyze the relationships between LIPUS-induced CCN2 and its repairing role in the meniscus.

Novel Cell Biological Assays for Measuring Bone Remodeling Activities of CCN Proteins.

Although two-dimensional (2D) cultures from bone lineage cells are often used, it is well-known that this culture system is completely different from the in vivo bone matrix environment. In this paper, we describe a 3D culture method using both the mouse osteocytic cell line, MLO-Y4, and an osteocyte-enriched population of the cells isolated from mice. These cells are embedded in collagen gel with recombinant cellular communication network (CCN) factor proteins; then, osteoblasts or osteoclasts are inoculated and cultured on the collagen gel. Because this method mimics the in vitro bone matrix environment, it is useful for understanding the detailed mechanism of actions of CCN proteins in the bone matrix.

Angiogenesis Assays for the Analysis of CCN Proteins.

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the actin of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) and CCN2-neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM). In addition, we introduce an autofluorescence imaging of blood vessels in the subcutaneous tumor xenograft mouse model. These assays can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

Mouse Models of Tumor Bone Metastasis and Invasion for Studying CCN Proteins.

Bone metastasis and bone destruction are common occurrences in human malignancies, including breast, prostate, and lung cancer, and are associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis and bone destruction are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the left cardiac ventricle. In addition, we introduce mouse model of the bone destruction by injection of SAS oral squamous cell carcinoma cells into the bone marrow space of the right tibial metaphysis. These assays can be applied to studies on roles of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) protein in tumor metastasis and development of treatment strategies targeting CCN proteins.

Elevated Expression of CCN3 in Articular Cartilage Induces Osteoarthritis in Hip Joints Irrespective of Age and Weight Bearing.

Osteoarthritis (OA) occurs not only in the knee but also in peripheral joints throughout the whole body. Previously, we have shown that the expression of cellular communication network factor 3 (CCN3), a matricellular protein, increases with age in knee articular cartilage, and the misexpression of CCN3 in cartilage induces senescence-associated secretory phenotype (SASP) factors, indicating that CCN3 promotes cartilage senescence. Here, we investigated the correlation between CCN3 expression and OA degenerative changes, principally in human femoral head cartilage. Human femoral heads obtained from patients who received total hip arthroplasty were categorized into OA and femoral neck fracture (normal) groups without significant age differences. Gene expression analysis of RNA obtained from femoral head cartilage revealed that CCN3 and MMP-13 expression in the non-weight-bearing part was significantly higher in the OA group than in the normal group, whereas the weight-bearing OA parts and normal cartilage showed no significant differences in the expression of these genes. The expression of COL10A1, however, was significantly higher in weight-bearing OA parts compared with normal weight-bearing parts, and was also higher in weight-bearing parts compared with non-weight-bearing parts in the OA group. In contrast, OA primary chondrocytes from weight-bearing parts showed higher expression of CCN3, p16, ADAMTS4, and IL-1ß than chondrocytes from the corresponding normal group, and higher ADAMTS4 and IL-1ß in the non-weight-bearing part compared with the corresponding normal group. Acan expression was significantly lower in the non-weight-bearing group in OA primary chondrocytes than in the corresponding normal chondrocytes. The expression level of CCN3 did not show significant differences between the weight-bearing part and non-weight-bearing part in both OA and normal primary chondrocytes. Immunohistochemical analysis showed accumulated CCN3 and aggrecan neoepitope staining in both the weight-bearing part and non-weight-bearing part in the OA group compared with the normal group. The CCN3 expression level in cartilage had a positive correlation with the Mankin score. X-ray analysis of cartilage-specific CCN3 overexpression mice (Tg) revealed deformation of the femoral and humeral head in the early stage, and immunohistochemical analysis showed accumulated aggrecan neoepitope staining as well as CCN3 staining and the roughening of the joint surface in Tg femoral and humeral heads. Primary chondrocytes from the Tg femoral head showed enhanced expression of Ccn3, Adamts5, p16, Il-6, and Tnfα, and decreased expression of Col2a1 and -an. These findings indicate a correlation between OA degenerative changes and the expression of CCN3, irrespective of age and mechanical loading. Furthermore, the Mankin score indicates that the expression level of Ccn3 correlates with the progression of OA.

Fibroblast Growth Factors and Cellular Communication Network Factors: Intimate Interplay by the Founding Members in Cartilage.

Fibroblast growth factors (FGFs) constitute a large family of signaling molecules that act in an autocrine/paracrine, endocrine, or intracrine manner, whereas the cellular communication network factors (CCN) family is composed of six members that manipulate extracellular signaling networks. FGFs and CCNs are structurally and functionally distinct, except for the common characteristics as matricellular proteins. Both play significant roles in the development of a variety of tissues and organs, including the skeletal system. In vertebrates, most of the skeletal parts are formed and grow through a process designated endochondral ossification, in which chondrocytes play the central role. The growth plate cartilage is the place where endochondral ossification occurs, and articular cartilage is left to support the locomotive function of joints. Several FGFs, including FGF-2, one of the founding members of this family, and all of the CCNs represented by CCN2, which is required for proper skeletal development, can be found therein. Research over a decade has revealed direct binding of CCN2 to FGFs and FGF receptors (FGFRs), which occasionally affect the biological outcome via FGF signaling. Moreover, a recent study uncovered an integrated regulation of FGF and CCN genes by FGF signaling. In this review, after a brief introduction of these two families, molecular and genetic interactions between CCN and FGF family members in cartilage, and their biological effects, are summarized. The molecular interplay represents the mutual involvement of the other in their molecular functions, leading to collaboration between CCN2 and FGFs during skeletal development.

Effect of Angiotensin II on Chondrocyte Degeneration and Protection via Differential Usage of Angiotensin II Receptors.

The renin-angiotensin system (RAS) controls not only systemic functions, such as blood pressure, but also local tissue-specific events. Previous studies have shown that angiotensin II receptor type 1 (AT1R) and type 2 (AT2R), two RAS components, are expressed in chondrocytes. However, the angiotensin II (ANG II) effects exerted through these receptors on chondrocyte metabolism are not fully understood. In this study, we investigated the effects of ANG II and AT1R blockade on chondrocyte proliferation and differentiation. Firstly, we observed that ANG II significantly suppressed cell proliferation and glycosaminoglycan content in rat chondrocytic RCS cells. Additionally, ANG II decreased CCN2, which is an anabolic factor for chondrocytes, via increased MMP9. In Agtr1a-deficient RCS cells generated by the CRISPR-Cas9 system, Ccn2 and Aggrecan (Acan) expression increased. Losartan, an AT1R antagonist, blocked the ANG II-induced decrease in CCN2 production and Acan expression in RCS cells. These findings suggest that AT1R blockade reduces ANG II-induced chondrocyte degeneration. Interestingly, AT1R-positive cells, which were localized on the surface of the articular cartilage of 7-month-old mice expanded throughout the articular cartilage with aging. These findings suggest that ANG II regulates age-related cartilage degeneration through the ANG II-AT1R axis.

RFX1-mediated CCN3 induction that may support chondrocyte survival under starved conditions.

Cellular communication network factor (CCN) family members are multifunctional matricellular proteins that manipulate and integrate extracellular signals. In our previous studies investigating the role of CCN family members in cellular metabolism, we found three members that might be under the regulation of energy metabolism. In this study, we confirmed that CCN2 and CCN3 are the only members that are tightly regulated by glycolysis in human chondrocytic cells. Interestingly, CCN3 was induced under a variety of impaired glycolytic conditions. This CCN3 induction was also observed in two breast cancer cell lines with a distinct phenotype, suggesting a basic role of CCN3 in cellular metabolism. Reporter gene assays indicated a transcriptional regulation mediated by an enhancer in the proximal promoter region. As a result of analyses in silico, we specified regulatory factor binding to the X-box 1 (RFX1) as a candidate that mediated the transcriptional activation by impaired glycolysis. Indeed, the inhibition of glycolysis induced the expression of RFX1, and RFX1 silencing nullified the CCN3 induction by impaired glycolysis. Subsequent experiments with an anti-CCN3 antibody indicated that CCN3 supported the survival of chondrocytes under impaired glycolysis. Consistent with these findings in vitro, abundant CCN3 production by chondrocytes in the deep zones of developing epiphysial cartilage, which are located far away from the synovial fluid, was confirmed in vivo. Our present study uncovered that RFX1 is the mediator that enables CCN3 induction upon cellular starvation, which may eventually assist chondrocytes in retaining their viability, even when there is an energy supply shortage.

Bipartite regulation of cellular communication network factor 2 and fibroblast growth factor 1 genes by fibroblast growth factor 1 through histone deacetylase 1 and fork head box protein A1.

Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities.

Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer.

Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones - CDC37, HSP90α and HSP90ß play key roles in cancer progression including epithelial-mesenchymal transition (EMT), although their contribution to EVs-mediated cell-cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer-derived EVs (MEV) were enriched with HSP90α HSP90ß and cancer-initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/ß reversed these MEV-driven malignancy events. In metastatic oral cancer patient-derived tumours, HSP90ß was significantly accumulated in infiltrating tumour-associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90-enriched MEV-induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA-mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90ß) could potentially be a novel therapeutic strategy to attenuate several EV-driven malignancy events in the tumour microenvironment. ABBREVIATIONS: CDC37: cell division control 37; EMT: epithelial-mesenchymal transmission; EV: extracellular vesicles; HNSCC: head and neck squamous cell carcinoma; HSP90: heat shock protein 90; TAM: tumour-associated macrophage.