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Shin-Ichi Hayashi

Publications and source records attributed to Shin-Ichi Hayashi.

At least 19 recordsLinked to original sources
PubMed2006

Constant rate of steady-state self-antigen trafficking from skin to regional lymph nodes.

It is suggested that dendritic cells (DCs) capture and present both foreign antigens such as components of pathogens as well as endogenous self-antigens. However, the magnitude of self-antigen trafficking to secondary lymphoid organs is still unclear. Here we show constitutive trafficking of self-antigens from skin to regional lymph nodes (LNs) quantitatively using a KRT14-Kitl transgenic mouse. This mouse model expresses the Kit ligand in keratinocytes, shows hyperpigmentation of the epidermis and exhibits constitutive accumulation of melanin granules (MGs) in skin regional LNs transported by Langerhans cells. Using an MG-solubilization technique, we revealed that 128 microg per week of MGs, a marker of self-antigens, accumulated in skin regional LNs and that the rate of accumulation was constant from 3 to 50 weeks. Activation markers such as CD40, CD54 and CD86 did not increase in the LNs, and abrogation of CD40 signaling did not affect the accumulation. Additionally, the total amount of MGs did not increase significantly following stimulation with intravenous LPS injections. These results suggest that the accumulation is not caused by inflammatory stimuli, and the steady-state trafficking of self-antigens is intrinsically maintained at a constant rate. Because the levels of self-antigens as well as the phenotype of these DCs are thought to be important in the strength of immune responses, the results may imply that the constant rate of trafficking of self-antigens is required for maintaining homeostatic conditions, such as self-tolerance.

Animals↗
PubMed2006

Potential of dental mesenchymal cells in developing teeth.

The tooth, composed of dentin and enamel, develops through epithelium-mesenchyme interactions. Neural crest (NC) cells contribute to the dental mesenchyme in the developing tooth and differentiate into dentin-secreting odontoblasts. NC cells are known to differentiate into chondrocytes and osteoblasts in the craniofacial region. However, it is not clear whether the dental mesenchymal cells in the developing tooth possess the potential to differentiate into a lineage(s) other than the odontoblast lineage. In this study, we prepared mesenchymal cells from E13.5 tooth germ cells and assessed their potential for differentiation in culture. They differentiated into odontoblasts, chondrocyte-like cells, and osteoblast-like cells. Their derivation was confirmed by tracing NC-derived cells as LacZ(+) cells using P0-Cre/Rosa26R mice. Using the flow cytometry-fluorescent di-beta-D-galactosidase system, which makes it possible to detect LacZ(+) cells as living cells, cell surface molecules of dental mesenchymal cells were characterized. Large number of LacZ(+) NC-derived cells expressed platelet-derived growth factor receptor alpha and integrins. Taken together, these results suggest that NC-derived cells with the potential to differentiate into chondrocyte-like and osteoblast-like cells are present in the developing tooth, and these cells may contribute to tooth organogenesis.

Animals↗
PubMed2006

Constitutively active beta-catenin promotes expansion of multipotent hematopoietic progenitors in culture.

This study was designed to investigate one component of the Wnt/beta-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable beta-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-Kit(low)Sca-1(low/-)CD19(-) CD11b/Mac-1(-)Flk-2(-)CD43(+)AA4.1(+)NK1.1(-)CD3(-)CD11c(-)Gr-1(-)CD45R/B220(+) cells in the presence of stromal cells and cytokines. They generated myeloid, T, and B lineage lymphoid cells in culture, but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term, stromal-free propagation, and a beta-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture, it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition, we demonstrate how it may be experimentally manipulated to generate valuable cell lines.

Animals↗
PubMed2006

Immunolocalization of liver receptor homologue-1 (LRH-1) in human breast carcinoma: possible regulator of insitu steroidogenesis.

Liver receptor homologue-1 (LRH-1) belongs to a class of nuclear orphan receptor. We examined immunolocalization of LRH-1 in 106 breast carcinomas. LRH-1 immunoreactivity was detected in 43% of the invasive ductal carcinoma. It was negatively correlated with clinical stage, histological grade and HER2 status, and positively associated with sex-steroid receptors, steroidogenic acute regulatory protein, P450 side-chain cleavage, and 3beta-hydroxysteroid dehydrogenase. LRH-1 immunoreactivity was also detected in 28% of the ductal carcinoma in situ. These results suggest that LRH-1 is frequently detected in breast carcinoma tissues, and plays important roles including the regulation of in situ steroidogenesis.

Breast Neoplasms↗
PubMed2005

Ascorbic acid promotes osteoclastogenesis from embryonic stem cells.

Ascorbic acid (AA) is known to regulate cell differentiation; however, the effects of AA on osteoclastogenesis, especially on its early stages, remain unclear. To examine the effects of AA throughout the process of osteoclast development, we established a culture system in which tartrate-resistant acid phosphate (TRAP)-positive osteoclasts were induced from embryonic stem cells without stromal cell lines. In this culture system, the number of TRAP-positive cells was strongly increased by the addition of AA during the development of osteoclast precursors, and reducing agents, 2-mercaptoethanol, monothioglycerol, and dithiothreitol, failed to substitute for AA. The effect of AA was stronger when it was added during the initial 4 days during the development of mesodermal cells than when it was added during the last 4 days. On day 4 of the culture period, AA increased the total cell recovery and frequency of osteoclast precursors. Magnetic cell sorting using anti-Flk-1 antibody enriched osteoclast precursors on day 4, and the proportion of Flk-1-positive cells but not that of platelet-derived growth factor receptor alpha-positive cells was increased by the addition of AA. These results suggest that AA might promote osteoclastogenesis of ES cells through increasing Flk-1-positive cells, which then give rise to osteoclast precursors.

Animals↗
PubMed2005

VEGF receptor 1 signaling is essential for osteoclast development and bone marrow formation in colony-stimulating factor 1-deficient mice.

VEGF receptor 1 (VEGFR-1/Flt-1) is a high-affinity tyrosine kinase (TK) receptor for VEGF and regulates angiogenesis as well as monocyte/macrophage functions. We previously showed that the osteoclast deficiency in osteopetrotic Csf1op/Csf1op (op/op) mice is gradually restored in an endogenous, VEGF-dependent manner. However, the molecular basis of the recovery is still not clear. To examine which VEGFR is important and to clarify how colony-stimulating factor 1 (CSF-1) and VEGF signals interact in osteoclastogenesis, we introduced a VEGFR-1 signaling deficiency (Flt1(TK)-/-) into op/op mice. The original Flt1(TK)-/- mice showed mild osteoclast reduction without bone marrow suppression. The double mutant (op/opFlt1(TK)-/-) mice, however, exhibited very severe osteoclast deficiency and did not have numbers of osteoclasts sufficient to form the bone marrow cavity. The narrow bone marrow cavity in the op/opFlt1(TK)-/- mice was gradually replaced with fibrous tissue, resulting in severe marrow hypoplasia and extramedullary hematopoiesis. In addition to osteoclasts, osteoblasts also decreased in number in the op/opFlt1(TK)-/- mice. These results strongly suggest that the interaction of signals by means of VEGFR-1 and the CSF-1 receptor plays a predominant role not only in osteoclastogenesis but also in the maintenance of bone marrow functions.

Animals↗
PubMed2005

Down-regulation of osteoprotegerin production in bone marrow macrophages by macrophage colony-stimulating factor.

Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) induce the differentiation of bone marrow macrophages (BMMs) into osteoclasts. To delineate mechanisms involved, the effect of M-CSF on the production of osteoprotegerin (OPG), decoy receptor of RANKL, in BMMs was investigated. Mouse bone marrow cells were cultured with M-CSF for 4 days and adherent cells formed were used as BMMs. BMMs were cultured with or without M-CSF, and analyzed for expression of OPG and receptor activator of NF-kappaB (RANK; receptor for RANKL) mRNAs by real-time polymerase chain reaction and secretion of OPG by enzyme-linked immunosorbent assay. BMMs expressed macrophage markers, CD115 (c-fms), Mac-1 and F4/80, and showed phagocytotic activity. In addition, BMMs expressed OPG mRNA and secreted OPG into medium. M-CSF inhibited both the OPG mRNA expression and the OPG secretion dose-dependently and reversibly. The expression of RANK mRNA was not significantly affected by M-CSF. The results showed that M-CSF suppresses the OPG production in BMMs, which may increase the sensitivity of BMMs to RANKL.

Animals↗
PubMed2005

Progesterone receptor in non-small cell lung cancer--a potent prognostic factor and possible target for endocrine therapy.

A possible involvement of gender-dependent factors has been postulated in development of human non-small-cell lung cancers (NSCLC), but its details remain unclear. In this study, we examined biological significance of progesterone receptor in NSCLCs. Progesterone receptor immunoreactivity was detected in 106 of 228 NSCLCs (46.5%). Progesterone receptor-positive NSCLC was frequently detected in female and adenocarcinoma, and was inversely associated with tumor-node-metastasis stage and histologic differentiation. Progesterone receptor status was also associated with better clinical outcome of the patients, and a multivariate analysis revealed progesterone receptor status as an independent prognostic factor. Progesterone-synthesizing enzymes were detected in NSCLCs, and tissue concentration of progesterone was higher in these cases (n = 42). Immunoblotting analyses showed the presence of progesterone receptor in three NSCLC cell lines (A549, LCSC#2, and 1-87), but not in RERF-LC-OK or PC3. Transcriptional activities of progesterone receptor were increased by progesterone in these three progesterone receptor-positive NSCLC cells by luciferase assays. Cell proliferation was inhibited by progesterone in these progesterone receptor-positive NSCLC cells in a dose-dependent manner, which was inhibited by progesterone receptor blocker. Proliferation of these tumor cells injected into nude mice was also dose-dependently inhibited by progesterone, with a concomitant increase of p21 and p27 and a decrease of cyclin A, cyclin E, and Ki67. Results of our present study suggested that progesterone receptor was a potent prognostic factor in NSCLCs and progesterone inhibited growth of progesterone receptor-positive NSCLC cells. Therefore, progesterone therapy may be clinically effective in suppressing development of progesterone receptor-positive NSCLC patients.

Adult↗
PubMed2005

Significance of HDAC6 regulation via estrogen signaling for cell motility and prognosis in estrogen receptor-positive breast cancer.

Histone deacetylase (HDAC) 6 is a subtype of the HDAC family; it deacetylates alpha-tubulin and increases cell motility. Here, we investigate the impact of an alteration of HDAC6 expression in estrogen receptor alpha (ER)-positive breast cancer MCF-7 cells, as we identified that HDAC6 is a novel estrogen-regulated gene. MCF-7 treated with estradiol showed increased expression of HDAC6 mRNA and protein and a four-fold increase in cell motility in a migration assay. Cell motility was increased to the same degree by stably transfecting the HDAC6 expression vector into MCF-7 cells. In both cases, the cells changed in appearance from their original round shape to an axon-extended shape, like a neuronal cell. This HDAC6 accumulation caused the deacetylation of alpha-tubulin. Either the selective estrogen receptor modulator tamoxifen (TAM) or the pure antiestrogen ICI 182,780 prevented estradiol-induced HDAC6 accumulation and deacetylation of alpha-tubulin, leading to reduced cell motility. Tubacin, an inhibitory molecule that binds to the tubulin deacetylation domain of HDAC6, also prevented estradiol-stimulated cell migration. Finally, we evaluated HDAC6 protein expression in 139 consecutively archived human breast cancer tissues by immunohistochemical staining. The prognostic analyses for these patients revealed no significant differences based on HDAC6 expression. However, subset analysis of ER-positive patients who received adjuvant treatment with TAM (n = 67) showed a statistically significant difference in relapse-free survival and overall survival in favor of the HDAC6-positive group (P < 0.02 and P < 0.05, respectively). HDAC6 expression was an independent prognostic indicator by multivariate analysis (odds ratio = 2.82, P = 0.047). These results indicate the biological significance of HDAC6 regulation via estrogen signaling.

Anilides↗
PubMed2005

Presence and distribution of neural crest-derived cells in the murine developing thymus and their potential for differentiation.

Neural crest (NC) cells are multipotent cells that can differentiate into melanocytes, neurons, glias and myofibroblasts. They migrate into the fetal thymus on embryonic day (E) 12 in mice and may participate in thymic organogenesis. Although the abnormality of migration and distribution of NC cells in the thymus results in immunodeficiency, the spatial and temporal presence of their progeny cells has not been defined in detail. In this study, we traced NC-derived cells based on the myelin protein zero gene promoter-Cre-mediated excision. We demonstrated that large numbers of NC-derived cells in the thymus were detected on E11.5 to E16.5 but rarely on E17.5. A colony formation assay of single thymic cells demonstrated that multipotent cells with the potential to differentiate into melanocytes, neurons and/or glias were present in the E14.5 and E15.5 but not in the E17.5 fetal thymus. Furthermore, we confirmed that these multipotent cells were NC-derived cells. Taken together, these findings imply that multipotent NC-derived cells are present in the developing thymus, but rarely in this organ at a later stage, suggesting that NC-derived cells may play roles in thymic organogenesis at an early embryonic stage.

Animals↗
PubMed2005

Estrogen signaling and prediction of endocrine therapy.

Estrogen plays an important role in the growth and progression of human breast cancer. Understanding the whole picture of estrogen signaling is a very important goal towards clarifying the biology of this disease. On the other hand, hormonal therapy for breast cancer has been progressing rapidly with the advent of drugs such as selective estrogen receptor (ER) modulators and aromatase inhibitors. Prediction of individual response to these hormonal therapies is becoming important for the management of breast cancer patients. To help address these basic and clinical issues, we are developing several new tools such as the focused microarray and the green fluorescent protein-reporter cell system. We first carried out expression profiling of approximately 10,000 genes in ER-positive breast cancer cells. Based on the results, estrogen-responsive genes (ERG) were selected and a custom-made cDNA microarray consisting of 200 genes from a narrowed-down subset was produced. Using this microarray, we investigated various aspects of estrogen signaling such as the effect of estrogen-antagonists on ERG expression profile and functional analysis of ERbeta and novel estrogen responsive gene EGR3. Furthermore, expression levels of several candidate genes selected from the custom-array contents were analyzed by real-time RT-PCR and immunohistochemistry using breast cancer tissues to determine novel predictive factors for responsiveness to hormone therapy in primary breast cancer patients. Expression of several genes, such as HDAC6, significantly correlated with disease-free and overall survival of patients treated with adjuvant tamoxifen therapy. We are currently developing a new tool for analyzing the effects of novel aromatase inhibitors in individual breast cancer patients using estrogen-responsive element-green fluorescent protein-indicator cells. We hope that these approaches may provide not only new clues for elucidation of estrogen-dependent growth mechanisms of cancer, but also clinical benefits to patients by assessment of individual responses to endocrine therapy.

Breast Neoplasms↗
PubMed2005

Enforced expression of PU.1 rescues osteoclastogenesis from embryonic stem cells lacking Tal-1.

Transcription factor T-cell acute lymphocytic leukemia 1 (Tal-1) is essential for the specification of hematopoietic development. Mice lacking Tal1 fail to generate any hematopoietic precursors. Using our co-culture system with stromal cells, we demonstrate that enforced expression of the transcription factor PU.1 under tetracycline control in Tal1-null embryonic stem (ES) cells rescues the development of osteoclasts and macrophage-like phagocytes. It was low efficiency compared with wild-type ES cells; other hematopoietic lineage cells of granulocytes, B cells, mast cells, megakaryocytes, and erythroid cells were not generated. Osteoclasts developed in this culture were multinucleated and competent for bone resorption. Their development depended on macrophage colony-stimulating factor and receptor activator of nuclear factor kappaB ligand. The majority of cells with the potential to differentiate into osteoclasts expressed fetal liver kinase 1 (Flk-1) and could be isolated using anti-Flk-1 antibody. These results suggest that the expression of PU.1 is a critical event for osteoclastogenesis and that Tal-1 may lie upstream of PU.1 in a regulatory hierarchy during osteoclastogenesis.

Basic Helix-Loop-Helix Proteins↗
PubMed2004

HDAC6 expression is correlated with better survival in breast cancer.

PURPOSE: The structure and function of chromatin can be altered by modifications to histone. Histone acetylation in vivo is a dynamic reversible process governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC6 is a unique isoform among the HDACs, and a gene expression pattern study, with cDNA microarray in MCF-7 cells, showed the HDAC6 gene to be late responsive, estrogen induced, and up-regulated. This led us to hypothesize that there was a link between levels of HDAC6 expression and the metastatic potential of breast cancer and also, therefore, the prognosis of these patients. EXPERIMENTAL DESIGN: In the present study, the level of HDAC6 mRNA expression was analyzed with quantitative real-time reverse transcription-PCR, in 135 female patients with invasive breast cancer. HDAC6 protein expression was also determined by immunohistochemistry. An association was sought between HDAC6 expression and various clinicopathologic factors. RESULTS: HDAC6 mRNA was expressed at significantly higher levels in breast cancer patients with small tumors measuring less than 2 cm, with low histologic grade, and in estrogen receptor alpha- and progesterone receptor-positive tumors. By contrast, no relationship was found between HDAC6 mRNA expression and any of the other clinicopathologic factors, namely, age, menopausal status, and axillary lymph node involvement. Patients expressing high levels of HDAC6 mRNA and protein had a better prognosis than those expressing low levels, in terms of disease-free survival. However, multivariate analysis failed to show that HDAC6 mRNA and protein are an independent prognostic factors for disease-free survival and overall survival. Furthermore, the patients with high levels of HDAC6 mRNA tended to be more responsive to endocrine treatment than those with low levels. Specific HDAC6 staining was found in the nucleus of some normal epithelial cells and in the cytoplasm of the majority of cancer cells. Although postmenopausal patients showed higher HDAC6 protein expression, there were no relationship between protein expression and any other clinicopathologic factors. CONCLUSIONS: We conclude that the levels of HDAC6 mRNA expression may have potential both as a marker of endocrine responsiveness and also as a prognostic indicator in breast cancer. Additional investigations are warranted concerning the relationship between HDAC6 expression and response to endocrine therapy.

Adult↗
PubMed2004

Lipopolysaccharide-induced osteoclastogenesis in Src homology 2-domain phosphatase-1-deficient viable motheaten mice.

Osteoclasts are hemopoietic cells that participate in bone resorption and remodeling. Receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are critical for development of osteoclasts. The Toll-like receptor (TLR) family shares some of the downstream signaling with RANK. The TLR4 ligand, lipopolysaccharide (LPS), is reported to accelerate bone lysis; however, signaling via TLRs has never been reported to induce osteoclastogenesis without RANKL. In this study we showed that significant numbers of mature osteoclasts were generated from protein tyrosine phosphatase Src homology 2-domain phosphatase-1-defective Hcph(me-v)/Hcph(me-v) (me(v)/me(v)) bone marrow cells in the presence of M-CSF and LPS without addition of RANKL in culture. This M-CSF plus LPS-induced osteoclastogenesis was not inhibited by an anti-TNFalpha antagonistic antibody or by osteoprotegerin, a decoy receptor for RANKL. The replacement of RANKL by TLR ligands only occurred with LPS. Other ligands, a peptidoglycan for TLR2 or an unmethylated CpG oligonucleotide for TLR9, did not support osteoclast generation. The osteoclast precursors as well as RANKL-responsive osteoclast precursors were present in the Kit-positive cell-enriched fraction of bone marrow cells. Although me(v)/me(v) bone marrow cells required a comparable concentration of RANKL or TNFalpha as wild-type cells for the initiation of osteoclastogenesis, the numbers of multinucleated osteoclasts in me(v)/me(v) bone marrow cultures were significantly increased by the equivalent dose of RANKL or TNFalpha in the presence of M-CSF. These results indicate that a defect of Src homology 2-domain phosphatase-1 function not only accelerates physiological osteoclast development by RANKL/RANK, but also acquires a novel pathway for osteoclastogenesis by LPS.

Animals↗
PubMed2004

Prediction of hormone sensitivity by DNA microarray.

Endocrine-therapy continues to be extensively developed for treatment of breast cancer, and accurate therapeutic prediction of this hormone-associated cancer is strongly desired. Moreover, the role of estrogen and its receptor on the estrogen-dependent growth of breast cancer cells has not been clarified hitherto. Thus, to develop a new diagnostic tool for endocrine-therapy, and to address the molecular mechanism of estrogen-dependent breast carcinogenesis, we investigated the gene expression profile of estrogen-responsive genes in breast cancer using DNA microarray technique. We first comprehensively analyzed the profile of estrogen responsiveness among several estrogen receptor (ER)-positive cancer cell lines by a large-scale DNA microarray. Based on the obtained information, a total of 138 genes which showed high induction or repression of the expression by estrogen stimulation were selected and provided for custom microarray. The results of the custom microarray analysis were consistent with those of large-scale microarray analysis, and revealed that they were clearly categorized into early- or late-response types. Further analysis of these genes may provide new clues in the elucidation of the estrogen-dependent growth mechanisms of cancer. Furthermore, the custom microarray analysis of ER-positive breast cancer tissues also showed similar but not identical profiles to those of cell lines, indicating the potential of this custom microarray to predict the response to endocrine-therapy in the breast cancer. Moreover, in order to discover the new predictive factors for endocrine therapy in breast cancer patients, several candidate genes were selected and their expressions in breast cancer tissues were analyzed by real-time RT-PCR and by immunohistochemical technique. These studies could provide new clues for elucidation of the estrogen-dependent mechanisms of cancer and clinical benefit for patients.

Breast Neoplasms↗
PubMed2003

Distinct osteoclast precursors in the bone marrow and extramedullary organs characterized by responsiveness to Toll-like receptor ligands and TNF-alpha.

Osteoclasts are derived from hemopoietic stem cells and play critical roles in bone resorption and remodeling. Multinucleated osteoclasts are attached tightly to bone matrix, whereas precursor cells with the potential to differentiate into osteoclasts in culture are widely distributed. In this study, we assessed the characteristics of osteoclast precursors in bone marrow (BM) and in extramedullary organs as indicated by their responsiveness to ligands for Toll-like receptors (TLRs) and to TNF-alpha. Development of osteoclasts from precursor cells in the BM was inhibited by CpG oligonucleotides, a ligand for TLR9, but not by LPS, a ligand for TLR4. BM osteoclasts were induced by TNF-alpha as well as receptor activator of NF-kappaB ligand in the presence of M-CSF. Splenic osteoclast precursors, even in osteoclast-deficient osteopetrotic mice, differentiated into mature osteoclasts following exposure to TNF-alpha or receptor activator of NF-kappaB ligand. However, splenic osteoclastogenesis was inhibited by both LPS and CpG. Osteoclastogenesis from peritoneal precursors was inhibited by not only these TLR ligands but also TNF-alpha. The effects of peptidoglycan, a ligand for TLR2, were similar to those of LPS. BM cells precultured with M-CSF were characterized with intermediate characteristics between those of splenic and peritoneal cavity precursors. Taken together, these findings demonstrate that osteoclast precursors are not identical in the tissues examined. To address the question of why mature osteoclasts occur only in association with bone, we may characterize not only the microenvironment for osteoclastogenesis, but also the osteoclast precursor itself in intramedullary and extramedullary tissues.

Animals↗
PubMed2003

Generation of structures formed by lens and retinal cells differentiating from embryonic stem cells.

Embryonic stem cells have the potential to give rise to all cell lineages when introduced into the early embryo. They also give rise to a limited number of different cell types in vitro in specialized culture systems. In this study, we established a culture system in which a structure consisting of lens, neural retina, and pigmented retina was efficiently induced from embryonic stem cells. Refractile cell masses containing lens and neural retina were surrounded by retinal pigment epithelium layers and, thus, designated as eye-like structures. Developmental processes required for eye development appear to proceed in this culture system, because the formation of the eye-like structures depended on the expression of Pax6, a key transcription factor for eye development. The present culture system opens up the possibility of examining early stages of eye development and also of producing cells for use in cellular therapy for various diseases of the eye.

Animals↗