Harumi Kawaki

Differential roles of CCN family proteins during osteoblast differentiation: Involvement of Smad and MAPK signaling pathways

CCN family proteins play diverse roles in many aspects of cellular processes such as proliferation, differentiation, adhesion, migration, angiogenesis and survival. In the bone tissue of vertebrate species, the expression of most CCN family members has been observed in osteoblasts. However, their spatial and temporal distributions, as well as their functions, are still only partially understood. In this study, we evaluated the localization of CCN family members in skeletal tissue in vivo and comparatively analyzed the gene expression patterns and functions of the members in murine osteoblasts in primary culture. Immunofluorescent analyses revealed that the CCN family members were differentially produced in osteoblasts and osteocytes. The presence of all Ccn transcripts was confirmed in those osteoblasts. Among the members, CCN1, CCN2, CCN4 and CCN5 were found in osteocytes. CCN4 and CCN5 were distributed in osteocytes located inside of bone matrix as well. Next, we investigated the expression pattern of Ccn family members during osteoblast differentiation. Along with differentiation, most of the members followed proper gene expression patterns; whereas, Ccn4 and Ccn5 showed quite similar patterns. Furthermore, we evaluated the effects of CCN family members on the osteoblastic activities by using recombinant CCN proteins and RNA interference method. Five members of this family displayed positive effects on osteoblast proliferation or differentiation. Of note, CCN3 drastically inhibited the osteoblast activities. Each Ccn specific siRNA could modulate osteoblast activities in a manner expected by the observed effect of respective recombinant CCN protein. In addition, we found that extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinase pathways were critically involved in the CCN family member-mediated modification of osteoblast activities. Collectively, all Ccn family members were found to be differentially expressed along with differentiation and therefore could participate in progression of the osteoblast lineage.

Role of Mechanical Stress-induced Glutamate Signaling-associated Molecules in Cytodifferentiation of Periodontal Ligament Cells

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.

Novel effects of CCN3 that may direct the differentiation of chondrocytes.

Identification and characterization of local molecules directing the differentiation of chondrocytes to either transient or permanent cartilage are major issues in cartilage biology. Here, we found CCN family protein 3 (CCN3) was abundantly produced in rat developing epiphyseal cartilage. Evaluations in vitro showed that CCN3 repressed epiphyseal chondrocyte proliferation, while promoting matrix production in multiple assays performed. Furthermore, CCN3 enhanced the articular chondrocytic phenotype; whereas it repressed the one representing endochondral ossification. Additionally, the phenotype of growth plate chondrocytes and chondrogenic progenitors also appeared to be affected by CCN3 in a similar manner. These findings suggest a significant role of CCN3 in inducing chondrocytes to articular ones during joint formation.

Compressive force-produced CCN2 induces osteocyte apoptosis through ERK1/2 pathway.

Osteocytes produce various factors that mediate the onset of bone formation and resorption and play roles in maintaining bone homeostasis and remodeling in response to mechanical stimuli. One such factor, CCN2, is thought to play a significant role in osteocyte responses to mechanical stimuli, but its function in osteocytes is not well understood. Here, we showed that CCN2 induces apoptosis in osteocytes under compressive force loading. Compressive force increased CCN2 gene expression and production, and induced apoptosis in osteocytes. Application of exogenous CCN2 protein induced apoptosis, and a neutralizing CCN2 antibody blocked loading‐induced apoptosis. We further examined how CCN2 induces loaded osteocyte apoptosis. In loaded osteocytes, extracellular signal‐regulated kinase 1/2 (ERK1/2) was activated, and an ERK1/2 inhibitor blocked loading‐induced apoptosis. Furthermore, application of exogenous CCN2 protein caused ERK1/2 activation, and the neutralizing CCN2 antibody inhibited loading‐induced ERK1/2 activation. Therefore, this study demonstrated for the first time to our knowledge that enhanced production of CCN2 in osteocytes under compressive force loading induces apoptosis through activation of ERK1/2 pathway.

CCN2 as a Novel Molecule Supporting Energy Metabolism of Chondrocytes

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 few genes 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. J. Cell. Biochem. 115: 854–865, 2014.

Mechanisms of the immunosuppressive effects of mouse adipose tissue-derived mesenchymal stromal cells on mouse alloreactively stimulated spleen cells

The mechanisms of immunomodulation by mesenchymal stromal cells remain poorly understood. In this study, the effects of mouse adipose tissue-derived mesenchymal stromal cells (ASCs) on mouse spleen cells alloreactively stimulated by anti-CD3 and anti-CD28 antibody-coated (anti-CD3/CD28) beads were observed. Production of interferon-γ by the anti-CD3/CD28 bead-stimulated spleen cells was significantly suppressed in co-culture with ASCs. However, an augmented intensity of CD69 on the stimulated spleen cells was not suppressed in the presence of ASCs. The immunosuppressive effects of ASCs were partially mediated by one or more soluble factors (26% suppression). However, the ASCs require cell-cell contact in order to maximally exert suppression (88%). The suppressive effect of ASCs mediated by direct cell contact was partially reversed following knockdown of β2 microglobulin, a component of the major histocompatibility complex (MHC) class I molecule, with siRNA. The results of the study demonstrated that ASCs have significant immune modulatory effects on alloreactively stimulated spleen cells. The effects of ASCs on spleen cells are dependent on soluble factor(s) and cell contact, which is mediated by the MHC class I complex on ASCs.

Association of the metastatic phenotype with CCN family members among breast and oral cancer cells.

The CCN family of proteins consists of six members with conserved structural features. These proteins play several roles in the physiology and pathology of cells. Among the pathological roles of the CCN family, one of the most important and controversial ones is their role in the expansion and metastasis of cancer. Up to now a number of reports have described the possible role of each CCN family member independently. In this study, we comprehensively analyzed the roles of all six CCN family members in cell growth, migration and invasion of breast cancer cells in vitro and in vivo. As a result, we found the CCN2/CCN3 ratio to be a parameter that is associated with the metastatic phenotype of breast cancer cells that are highly metastatic to the bone. The same analysis with cell lines from oral squamous carcinomas that are not metastatic to the bone further supported our notion. These results suggest the functional significance of the interplay between CCN family members in regulating the phenotype of cancer cells.

Tumor growth limited to subcutaneous site vs tumor growth in pulmonary site exhibit differential effects on systemic immunities

To evaluate systemic immunity associated with tumor growth limited to a subcutaneous site versus growth proceeding at multiple tumor sites, we established syngeneic mouse subcutaneous and pulmonary tumor models by local subcutaneous and intravenous injection of colon carcinoma CT26 cells. We found that splenic myeloid-derived suppressor cell (MDSC) levels were significantly increased in the subcutaneous tumor model but not in the pulmonary tumor model. Furthermore, both CD4+ and CD8+ Txa0cells as well as CD4+ Foxp3+ Txa0cells were significantly decreased in the subcutaneous tumor model and were largely unchanged in the pulmonary tumor model. In addition, the subcutaneous model, but not the pulmonary model, displayed a Th1 polarization bias. This bias was characterized by decreased IL-4, IL-9, and IL-10 production, whereas the pulmonary model displayed increased production of IL-10. These results suggest that the mode of tumor development has differential effects on systemic immunity that may, in turn, influence approaches to treatment of cancer patients.

Role of CCN2 in Amino Acid Metabolism of Chondrocytes

CCN2/connective tissue growth factor (CTGF) is a multi‐functional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2‐null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2‐null and wild‐type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2‐null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2‐null chondrocytes. Furthermore, we newly found that expression of two genes encoding two glycolytic enzymes, as well as the previously reported Eno1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2‐null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro. Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage. J. Cell. Biochem. 117: 927–937, 2016.

Producing Capabilities of Interferon-gamma and Interleukin-10 in Peripheral Blood from Oral Squamous Cell Carcinoma Patients

In order to evaluate the Th1 and Th2 responses of Oral Squamous Cell Carcinoma (OSCC) patients, we investigated the cytokine producing capability of peripheral blood (PB), and compared it with clinicopathological appearances of OSCC patients. The production of a Th1-type cytokine, interferon (IFN)-γ, from lipopolysaccharide (LPS)-stimulated PB correlated positively with the frequency of lymph node metastasis. We also investigated the production of a Th2-type cytokine, IL-10, however, no significant correlation was observed with the clinicopathological appearances. Our results suggested that the IFN-γ producing capability was specifically regulated and dependent on the regional metastatic potencies of OSCCs.