Masako Akiyama

The effects of polyunsaturated fatty acids and their metabolites on osteoclastogenesis in vitro

Bone homeostasis is maintained by active remodeling through the balance between resorption (by osteoclasts) and synthesis (by osteoblasts). In this study, we examined the effects of polyunsaturated fatty acids (PUFAs) and their metabolites on sRANKL-induced differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts in vitro. Docosahexaenoic acid (DHA) strongly inhibited osteoclastogenesis; however, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) enhanced it. The enhancement effect of PUFAs on osteoclastogenesis was mediated predominantly by cyclooxygenase (COX) products, because the effect was inhibited by a COX inhibitor. It was also found that COX products of PUFAs, prostaglandin E(1), E(2), and E(3), clearly increased in osteoclastogenesis. The inhibitory effect of DHA on osteoclastogenesis was reversed by treatment with a lipoxygenase (LOX) inhibitor. Furthermore, resolvin D1, a LOX product of DHA, significantly inhibited osteoclastogenesis. Quantitative analysis of specific mRNA levels revealed that DHA-mediated attenuation of osteoclastogenesis might be due to a decrease in DC-STAMP expression. These results suggested that the effect of DHA on osteoclastogenesis is, at least in part, mediated by lipoxygenase products. This study showed a distinct mechanism of the effect of PUFAs on osteoclastogenesis and will provide evidence for therapeutic treatment with DHA in osteoporotic patients.

Communication-dependent mineralization of osteoblasts via gap junctions

Connexin43 (Cx43) is a major gap junction (GJ) protein in bone and plays a critical role in osteoblast differentiation. Several studies show that osteoblast differentiation is delayed by Cx43 ablation. However, the precise mechanism underlying the role of Cx43 in osteoblast differentiation is not fully understood. Firstly, we analyzed the phenotype of a conditional knockout mouse, which was generated by mating of an osterix promoter-driven Cre expressing mouse with a Cx43-floxed mouse. As expected, delayed ossification was observed. Secondly, we demonstrated that the cell communication via gap junctions played an important role in osteoblast differentiation using a tamoxifen-inducible knockout system in vitro. Genetic ablation of Cx43 resulted in both the disruption of cell-communications and the attenuation of osteoblast mineralization induced by BMP-2, but not by ascorbic acid. Moreover, restoring full-length Cx43 (382aa) expression rescued the impairment of osteoblast cell-communication and osteoblast mineralization; however, the expression of the Cx43 N-terminal mutant (382aaG2V) did not rescue either of them. Comparing the gene expression profiles, the genes directly regulated by BMP-2 were attenuated by Cx43 gene ablation. These results suggested that the cell-communication mediated by gap junctions was indispensable for normal differentiation of osteoblast induced by BMP-2.

Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment.

Effects of xenon irradiation of the stellate ganglion region on fibromyalgia

[Purpose] The aim of the study was to determine the effect of xenon irradiation of the stellate ganglion region on fibromyalgia. [Subjects] The study included 5 men and 22 women (age, 56.4 ± 16.3 years [range, 25–84 years]) who were diagnosed with fibromyalgia according to the modified 2010 criteria of the American College of Rheumatology between July and August 2013. [Methods] Bilateral xenon light irradiation (0.38–1.1 μm) around the stellate ganglion was performed in the supine position by physical therapists using a xenon phototherapy device. We evaluated pain before and after irradiation using the visual analogue scale. [Results] We did not observe a relationship between the change in the visual analogue scale score and duration of fibromyalgia. However, we observed a relationship between the change in the visual analogue scale score and the score for the Japanese version of the Fibromyalgia Impact Questionnaire using the Cochran-Armitage test for trend. [Conclusion] Xenon light irradiation of the stellate ganglion significantly decreased the visual analogue scale score in patients with fibromyalgia having a higher score in the Fibromyalgia Impact Questionnaire, suggesting that a stronger effect could be obtained in patients with more severe fibromyalgia.

Impact of Docosahexaenoic Acid on Gene Expression during Osteoclastogenesis in Vitro—A Comprehensive Analysis

Polyunsaturated fatty acids (PUFAs), especially n-3 polyunsaturated fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are known to protect against inflammation-induced bone loss in chronic inflammatory diseases, such as rheumatoid arthritis, periodontitis and osteoporosis. We previously reported that DHA, not EPA, inhibited osteoclastogenesis induced by the receptor activator of nuclear factor-κB ligand (sRANKL) in vitro. In this study, we performed gene expression analysis using microarrays to identify genes affected by the DHA treatment during osteoclastogenesis. DHA strongly inhibited osteoclastogenesis at the late stage. Among the genes upregulated by the sRANKL treatment, 4779 genes were downregulated by DHA and upregulated by the EPA treatment. Gene ontology analysis identified sets of genes related to cell motility, cell adhesion, cell-cell signaling and cell morphogenesis. Quantitative PCR analysis confirmed that DC-STAMP, an essential gene for the cell fusion process in osteoclastogenesis, and other osteoclast-related genes, such as Siglec-15, Tspan7 and Mst1r, were inhibited by DHA.

Linear measurement accuracy of dental CT images obtained by 64-slice multidetector row CT: the effects of mandibular positioning and pitch factor at CT scanning.

Purpose:To evaluate whether the measurement accuracy of dental CT images is affected by the mandibular positioning and the pitch factor at CT scanning. Materials and Methods:Three dry mandibles were scanned by 64-slice multidetector row CT (MDCT) and micro-CT. For MDCT scanning, 7 different mandibular positioning and 3 different pitch factors were applied. After reformatting dental CT images, the bone height was measured on these images. It was also measured on the corresponding micro-CT image, which was defined as the actual value. The difference of the measurement values between these 2 was defined as the measurement error. Results:There was no significant difference in the measurement errors due to either the mandibular positioning or the pitch factor. Conclusion:The measurement accuracy of dental CT images obtained was not affected by either mandibular positioning or pitch factor at CT scanning.

Role of intercellular adhesion molecule-2 in osteoclastogenesis

Osteoclasts, multinucleated bone‐resorbing cells, are specialized cells derived from the monocyte/macrophage lineage. Therefore, it is essential for mononuclear precursors to find a fusion partner during its differentiation. Our previous study showed an important role of cell communication via Mac‐1 (CD11b/CD18) during osteoclastogenesis. However, the counter receptor of Mac‐1 was still unknown. Flow cytometric analysis showed that bone marrow‐derived mononuclear cells, used as osteoclast precursors, expressed intercellular adhesion molecule‐1 and ‐2. Quantitative RT‐PCR analysis revealed that expression level of ICAM‐2 was higher than that of ICAM‐1 in bone marrow cells. The osteoclastogenesis induced by receptor activator of NF‐kappaB ligand (RANKL) was inhibited by anti‐ICAM‐2 neutralizing antibody but not by anti‐ICAM‐1 neutralizing antibody. The inhibitory effect of anti‐ICAM‐2 antibody on osteoclastogenesis was enhanced by simultaneous treatment of anti‐CD11b neutralizing antibody. Furthermore, osteoclastogenesis induced by tumor necrosis factor α (TNFα) was also inhibited by anti‐ICAM‐2 neutralizing antibody. The involvement of lymphocytes in osteoclastogenesis was excluded, because anti‐ICAM‐2 antibody inhibited osteoclastogenesis using bone marrow‐derived cells from immunodeficiency mice. Immunocytochemical staining demonstrated colocalization of ICAM‐2 and Mac‐1 during osteoclastogenesis; however, Mac‐1 immunoreactivity was lost in differentiated multinucleated osteoclast. These results suggest the important role of ICAM‐2/Mac‐1 binding in osteoclastogenesis induced by either RANKL or TNFα.

Involvement of CX3CL1 in the Migration of Osteoclast Precursors Across Osteoblast Layer Stimulated by Interleukin-1ß.

The trigger for bone remodeling is bone resorption by osteoclasts. Osteoclast differentiation only occurs on the old bone, which needs to be repaired under physiological conditions. However, uncontrolled bone resorption is often observed in pro‐inflammatory bone diseases, such as rheumatoid arthritis. Mature osteoclasts are multinuclear cells that differentiate from monocyte/macrophage lineage cells by cell fusion. Although Osteoclast precursors should migrate across osteoblast layer to reach bone matrix before maturation, the underlying mechanisms have not yet been elucidated in detail. We herein found that osteoclast precursors utilize two routes to migrate across osteoblast layer by confocal‐ and electro‐microscopic observations. The osteoclast supporting activity of osteoblasts inversely correlated with osteoblast density and was positively related to the number of osteoclast precursors under the osteoblast layer. Osteoclast differentiation was induced by IL‐1ß, but not by PGE2 in high‐density osteoblasts. Osteoblasts and osteoclast precursors expressed CX3CL1 and CX3CR1, respectively, and the expression of CX3CL1 increased in response to interleukin‐1ß. An anti‐CX3CL1‐neutralizing antibody inhibited the migration of osteoclast precursors and osteoclast differentiation. These results strongly suggest the involvement of CX3CL1 in the migration of osteoclast precursors and osteoclastogenesis, and will contribute to the development of new therapies for bone diseases. J. Cell. Physiol. 232: 1739–1745, 2017.

A novel mode of stimulating platelet formation activity in megakaryocytes with peanut skin extract

We report in this study novel biochemical activities of peanut skin extract (PEXT) on thrombocytopoiesis. Peanut skin, derived from Arachis hypogaea L., is a traditional Chinese medicine that is used to treat chronic hemorrhage. We have shown that oral administration of PEXT increases the peripheral platelet levels in mice. Recently, we reported a liquid culture system that is useful for investigating megakaryocytopoiesis and thrombocytopoiesis from human CD34+ cells. In this liquid culture system, PEXT was shown to enhance the formation of CD41+/DAPI− cells (platelets), but had no effect on the formation of CD41+/DAPI+ cells (megakaryocytes) or on the DNA content. Furthermore, PEXT selectively stimulated proplatelet formation from cultured mature megakaryocytes and phorbol 12-myristate 13 acetate (PMA)-induced formation of platelet-like particles from Meg01 cells. Despite having no influence on the formation of megakaryocyte colony forming units (CFUs), PEXT increased the size of megakaryocytes during their development from CD34+ cells. PEXT showed no effect on the GATA-1 and NF-E2 mRNA levels, which are known to play an important role in thrombocytopoiesis and, based on the results of a pMARE-Luc (pGL3-MARE-luciferase) assay, had no influence on NF-E2 activation in Meg01 cells. These results suggest that PEXT accelerates proplatelet formation from megakaryocytes but does not influence the development of hematopoietic stem cells into megakaryocytes.

Volumetric changes in transplanted vascularized fat flaps after ischemic or congestive stresses and their relationship to capillary density in a zucker fatty rat model.

The aim of this study was to examine volumetric changes in fat flaps after stresses as well as their relationship with capillary density (CD) in a Zucker fatty rat model.