Yoshiko Shiratsuchi

Expression of Integrin β3 Is Correlated to the Properties of Quiescent Hemopoietic Stem Cells Possessing the Side Population Phenotype

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high expression of integrin β3 (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin β3 is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineage− (Lin−)/integrin β3high (β3high) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit+/Sca-1+/Lin− SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin β3 subunit that is present in SP cells having the properties of HSCs, is associated with integrin αv (CD51). Therefore, our results demonstrate that high expression of integrin β3 is correlated to the properties of quiescent HSCs and suggest that the integrin β3 subunit is available as a common surface marker of tissue-specific stem cells.

Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis

TNF-alpha, IFN-gamma, IL-4, and MIP-2 are known to be involved in Con A-induced hepatitis. Although Kupffer cells are reportedly involved in TNF-alpha production, it is largely unknown whether or not Kupffer cells also play a role in the production of other cytokines, such as IFN-gamma, IL-4, and MIP-2. In this study we examined the liver injury and the levels of plasma cytokines, including above four cytokines, KC, and IL-10 in Kupffer cell-depleted mice obtained through administration of liposome-encapsulated dichloromethylene bisphosphonate. The liver injury was significantly suppressed in Kupffer cell-depleted mice, as assessed as to the plasma ALT level and histochemistry. The cytokine levels were also significantly suppressed in such mice except for those of IFN-gamma, which was slightly suppressed at 12h, and IL-10, which was not significantly suppressed at any time. Apoptosis was also significantly suppressed in such mice, as found immunohistochemically with anti-ssDNA Ab. Taken together, these results suggest that Kupffer cells are involved in the production of MIP-2, KC, IL-4, and TNF-alpha in Con A-induced hepatitis, thereby contributing to the liver injury either directly or indirectly.

Infiltrating neutrophils induce allospecific CTL in response to immunization with apoptotic cells via MCP-1 production

Our previous studies demonstrated that i.p. injection of late apoptotic P388 cells caused phagocytosis by macrophages and transient infiltration of neutrophils into the peritoneal cavity. As neutrophils are known to function as effectors as well as regulators in the immune response, we examined the roles of infiltrating neutrophils in alloantigen‐specific CTL induction after immunization with late apoptotic P388 cells. The CTL induction and infiltration of CD8+ T cells into the peritoneal cavity were inhibited by depletion of neutrophils by anti‐Gr‐1 mAb or inhibition of neutrophil infiltration by anti‐MIP‐2 antibody, suggesting that neutrophils are involved in CD8+ T cell infiltration into the peritoneal cavity. It is known that MIP‐1α, MIP‐1β, and MCP‐1 are capable of attracting CD8+ T cells and that they are produced by neutrophils. These chemokines were detected in the peritoneal cavity, and among them, MCP‐1 production was reduced remarkably by suppression of neutrophil infiltration. Moreover, infiltration of CD8+ T cells into the peritoneal cavity as well as CTL activity was clearly reduced by administering anti‐MCP‐1 antibody i.p. Furthermore, the CTL induction and infiltration of CD8+ T cells in neutrophil‐depleted mice were restored significantly by administering recombinant murine MCP‐1 into the peritoneal cavity. These results indicate that MCP‐1 appears to link infiltration of neutrophils with CTL induction.

Nov/CCN3 regulates long-term repopulating activity of murine hematopoietic stem cells via integrin αvβ3

Throughout life, hematopoietic stem cells (HSCs) sustain the blood cell supply through their capacities for self-renewal and multilineage differentiation. These processes are regulated within a specialized microenvironment termed the ‘niche’. Here, we show a novel mechanism for regulating HSC function that is mediated by nephroblastoma overexpressed (Nov/CCN3), a matricellular protein member of the CCN family. We found that Nov contributes to the maintenance of long-term repopulating (LTR) activity through association with integrin αvβ3 on HSCs. The resultant β3 integrin outside-in signaling is dependent on thrombopoietin (TPO), a crucial cytokine involved in HSC maintenance. TPO was required for Nov binding to integrin αvβ3, and stimulated Nov expression in HSCs. However, in the presence of IFNγ, a cytokine known to impair HSC function, not only was TPO-induced expression of Nov suppressed, but the LTR activity was conversely impaired by TPO-mediated ligation of integrin αvβ3 with exogenous ligands, including Nov, as well. Thus, Nov/integrin αvβ3-mediated maintenance of HSCs appears to be modulated by simultaneous stimulation by other cytokines. Our finding suggests that this system contributes to the regulation of HSCs within the bone marrow niche.

Computational promoter modeling identifies the modes of transcriptional regulation in hematopoietic stem cells.

Extrinsic and intrinsic regulators are responsible for the tight control of hematopoietic stem cells (HSCs), which differentiate into all blood cell lineages. To understand the fundamental basis of HSC biology, we focused on differentially expressed genes (DEGs) in long-term and short-term HSCs, which are closely related in terms of cell development but substantially differ in their stem cell capacity. To analyze the transcriptional regulation of the DEGs identified in the novel transcriptome profiles obtained by our RNA-seq analysis, we developed a computational method to model the linear relationship between gene expression and the features of putative regulatory elements. The transcriptional regulation modes characterized here suggest the importance of transcription factors (TFs) that are expressed at steady state or at low levels. Remarkably, we found that 24 differentially expressed TFs targeting 21 putative TF-binding sites contributed significantly to transcriptional regulation. These TFs tended to be modulated by other nondifferentially expressed TFs, suggesting that HSCs can achieve flexible and rapid responses via the control of nondifferentially expressed TFs through a highly complex regulatory network. Our novel transcriptome profiles and new method are powerful tools for studying the mechanistic basis of cell fate decisions.

Promotion of mouse ameloblast proliferation by Lgr5 mediated integrin signaling

Rodent incisors grow throughout the animals lives, and the tooth‐forming cells are provided from proximal ends of the incisors where the tooth epithelium forms a stem cell niche called cervical loop. The committing cells in a cervical loop actively begin to proliferate (pre‐ameloblasts), and differentiating into ameloblasts. This study showed that the lower incisors of mice null for CD61 (CD61−/−), also known as integrin β3, were significantly shorter than those of the wild‐type mice at 8‐week‐old. The protein and mRNA expressions levels of Fgfr2, Lgr5, and Notch1, which are known to be involved in pre‐ameloblastic cell proliferation and stem cell maintenance, were reduced in the cervical loop of 2‐week‐old CD61−/− mice. The proliferation of pre‐ameloblasts was reduced in CD61−/− ameloblasts. The siRNA‐mediated suppression of CD61 (siCD61) reduced the proliferation of pre‐ameloblastic cell line ALC, and the expression levels of Lgr5 and Notch1 were reduced by the transfection with siCD61. The suppression of Lgr5 by transfection with siLgr5 suppressed the proliferation of the ALC cells. These results suggested that CD61 signaling is required for the proper growth of the cervical loop and for the promotion of the proliferation of pre‐ameloblastic cells through Lgr5. J. Cell. Biochem. 114: 2138–2147, 2013.

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.

The importance of infiltrating neutrophils in SDF-1 production leading to regeneration of the thymus after whole-body X-irradiation.

Our previous study demonstrated that neutrophils transiently infiltrated into a site where apoptosis had been induced. However, the role of infiltrating neutrophils has not been fully elucidated. In this study, we examined their role in regeneration of the thymus after whole-body X-irradiation by focusing on SDF-1 production. After X-irradiation, the thymus became severely atrophied presumably due to phagocytosis of apoptotic thymocytes. At that time, a significant number of neutrophils were detected in the thymus. The thymus was then partially regenerated on day 7, whereas the level of SDF-1 in it was significantly increased on days 3 and 5. Depletion of neutrophils greatly impaired SDF-1 production and the thymus regeneration. Moreover, administration of a CXCR4 antagonist also greatly suppressed the thymus regeneration. Furthermore, coculturing of a stromal cell line with infiltrating neutrophils increased SDF-1 production. These results suggest that infiltrating neutrophils play an auxiliary role in regeneration of the thymus after whole-body X-irradiation through augmentation of SDF-1 production.