Taeko Ishii

RANKL‐Induced Expression of Tetraspanin CD9 in Lipid Raft Membrane Microdomain Is Essential for Cell Fusion During Osteoclastogenesis

We showed that CD9, a member of tetraspanin superfamily proteins, is expressed in a specific membrane microdomain, called “lipid raft,” and is crucial for cell fusion during osteoclastogenesis after activation of the RANK/RANKL system.

Enhanced production of osteopontin in multiple myeloma: clinical and pathogenic implications

Summary. In this study, we examined osteopontin (OPN) production in myeloma cells and plasma OPN levels in multiple myeloma (MM) patients. We assessed OPN production in bone marrow cells (BMCs) by immunocytochemistry and enzyme‐linked immunosorbent assay (ELISA). We also assessed OPN production in various B‐cell malignant cell lines, including three myeloma cell lines by reverse transcription polymerase chain reaction (RT‐PCR) and Western blotting. In addition, we measured plasma OPN concentrations by ELISA in 30 MM patients, 21 monoclonal gammopathy of undetermined significance (MGUS) patients and 30 healthy volunteers. As a result, in an immunocytochemical study, abundant OPN was detected in BMCs from overt MM patients, whereas no OPN was detected in BMCs from patients with other haematological diseases, including MGUS. Cultured BMCs from overt MM patients produced more OPN than those from patients with either smouldering MM or MGUS. Myeloma cell lines spontaneously produced OPN. Plasma OPN levels of MM patients were significantly higher than those of MGUS patients and healthy volunteers (P < 0·05). Moreover, they correlated with both progression and bone destruction of the disease (P < 0·05). These suggest that myeloma cells actively produce OPN, which possibly contributes to osteoclastic bone resorption in MM. Plasma OPN levels may be a useful biomarker for assessing bone destruction in MM and distinguishing MM from MGUS or smouldering MM.

Tocilizumab, a humanized anti-interleukin-6 receptor antibody, ameliorated clinical symptoms and MRI findings of a patient with ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory osteoarticular disease. Although the etiology remains unknown, proinflammatory cytokines, such as tumor necrosis factor α and interleukin-6, have been implicated in the development of AS. Here, we report that a patient with AS, whose disease had been refractory to conventional treatment regimens and who needed to receive continuous corticosteroid, responded well to tocilizumab. While further clinical evaluation is required, tocilizumab may be an optional treatment for AS.

Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.

Intravital two-photon imaging: a versatile tool for dissecting the immune system.

During the past decade, multi-photon or ‘two-photon’ excitation microscopy has launched a new era in the field of biological imaging. The near-infrared excitation laser for two-photon microscopy can penetrate thicker specimens, enabling the visualisation of living cell behaviour deep within tissues and organs without thin sectioning. The minimised photobleaching and toxicity enables the visualisation of live and intact specimens for extended periods. In this brief review, recent findings in intravital two-photon imaging for the physiology and pathology of the immune system are discussed. The immune system configures highly dynamic networks, where many cell types actively travel throughout the body and interact with each other in specific areas. Hence, real-time intravital imaging may be a powerful tool for dissecting the mechanisms of this dynamic system. The most unique characteristic of the immune system is its highly dynamic nature. A variety of cell types, such as lymphocytes, macrophages and dendritic cells (DCs), are continuously circulating throughout the body, migrating through the peripheral tissues and interacting with each other in their respective niches. Conventional methodologies in immunology, such as flow cytometry, cell or tissue culture, biochemistry and histology, have brought tremendous achievement within this field, although the dynamics of immune cells in an entire animal remain less clear. Technological progress of fluorescence microscopy has enabled us to visualise the intact biological phenomenon that has been uninvestigated. Among the advancements, the recent emergence and prevalence of two-photon, excitation-based, laser microscopy has revolutionised the research field, such that the dynamic behaviour of cells deep inside living organs can be visualised and analysed.

The Role of Sphingosine 1-Phosphate in Migration of Osteoclast Precursors; an Application of Intravital Two-Photon Microscopy

Sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. Through intravital multiphoton imaging of bone tissues, we reveal that the bidirectional function of S1P temporospatially regulates the migration of osteoclast precursors within intact bone tissues. Imaging technologies have enabled in situ visualization of the behaviors of several players in intact tissues. In addition, intravital microscopy has the potential to be more widely applied to functional analysis and intervention.

Interferon-α acts on the S/G2/M phases to induce apoptosis in the G1 phase of an IFNAR2-expressing hepatocellular carcinoma cell line.

Background: The mode of action of interferon-α has been unknown. Results: Its point of action in the cell cycle was analyzed by single cell tracking using time lapse confocal imaging. Conclusion: Interferon-α activates p63 in S/G2/M and induces apoptosis and cell cycle arrest in the subsequent G1. Significance: Tracking cell cycle progression is crucial for understanding the mechanisms of interferon-α. Interferon-α (IFN-α) is used clinically to treat hepatocellular carcinoma (HCC), although the detailed therapeutic mechanisms remain elusive. In particular, IFN-α has long been implicated in control of the cell cycle, but its actual point of action has not been clarified. Here, using time lapse imaging analyses of the human HCC cell line HuH7 carrying a fluorescence ubiquitination-based cell cycle indicator (Fucci), we found that IFN-α induced cell cycle arrest in the G0/G1 phases, leading to apoptosis through an IFN-α type-2 receptor (IFNAR2)-dependent signaling pathway. Detailed analyses by time lapse imaging and biochemical assays demonstrated that the IFN-α/IFNAR2 axis sensitizes cells to apoptosis in the S/G2/M phases in preparation for cell death in the G0/G1 phases. In summary, this study is the first to demonstrate the detailed mechanism of IFN-α as an anticancer drug, using Fucci-based time lapse imaging, which will be informative for treating HCC with IFN-α in clinical practice.

Successful treatment of acquired hemophilia A, complicated by chronic GVHD, with tocilizumab

A 65-year-old woman who had suffered from chronic graft-versus-host disease (GVHD) presented with extensive purpura and was diagnosed with acquired hemophilia A. Because she was refractory to corticosteroids and her condition was complicated with diabetes mellitus, glaucoma, and hypoglobulinemia, she was treated with tocilizumab. Tocilizumab treatment increased the activity of factor VIII in a rapid and sustained manner, leading to a reduction of the prednisolone dose. Tocilizumab may thus be an optional treatment modality for acquired hemophilia A.

Control of osteoclast precursor migration: A novel point of control for osteoclastogenesis and bone homeostasis

Osteoclasts are bone-resorbing, multinucleated giant cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. They have critical roles not only in normal bone remodeling but also during pathogenesis of destructive bone disorders such as osteoporosis, rheumatoid arthritis, and cancers metastatic to bone. Many molecules, especially macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL), make significant contributions to osteoclast differentiation. However, the process of osteoclast precursor trafficking to and from the bone surface, where cell fusion occurs to form the fully differentiated multinucleated cells that mediate bone resorption, is less well-documented. Recent studies have shed light on the mechanisms involved and have demonstrated the vital participation of various chemokines such as CCL2, CCL5, CXCL12, and CX3CL1, and lipid mediators such as sphingosine-1-phosphate (S1P). In addition, advances in imaging technologies, such as the development of intravital multiphoton microscopy, have enabled the in situ visualization of the behavior of osteoclasts and their precursors within intact bone tissue. This capability will be extremely useful for dissecting the mechanisms controlling the migration of these cells in vivo. In this Perspective, we review the latest knowledge in this new field of bone biology, with a focus on novel imaging methodology and its applications in this field. IBMS BoneKEy. 2010 August;7(8):279-286. ©2010 International Bone & Mineral Society

A case of multicentric Castleman's disease demonstrating severe eosinophilia and enhanced production of interleukin-5.

Abstract: Introduction: Castlemans disease (CD), idiopathic lymph‐node hyperplasia, is a heterogeneous disease of unknown origin. Although the pathophysiology is yet to be elucidated, interleukin (IL)‐6 produced by swollen lymph nodes has been reported to play a crucial role in CD.