Epigenetic regulation of the neuroblastoma genes, Arid3b and Mycn.

AT-rich interaction domain molecule 3B (ARID3B) and MYCN are expressed in a portion of neuroblastoma, and form a combination that has strong oncogenic activity in mouse embryonic fibroblasts (MEFs). Here, we show that this combination can also convert neural stem cells to neuroblastoma-like tumor. To address whether there are common mechanisms regulating the expression of this combination of genes, we examined public repositories of gene expression data and found that although these genes are rarely expressed together, co-expression was observed in a proportion of germ cell tumors (GCTs), in embryonic stem (ES) cells and in testis. These cell types and tissues are related to pluripotency and we show here that in mouse ES cells, Arid3b and Mycn are indeed involved in cell proliferation; the former in avoiding cell death and the latter in driving cell cycle progression. Accordingly, the two genes are induced during somatic cell reprogramming to iPS, and this induction is accompanied by the switching of promoter histone marks from H3K27me3 to H3K4me3. Conversely, the switch from H3K4me3 to H3K27me3 in these genes occurs during the differentiation of neural crest to mature sympathetic ganglia cells. In many, if not most, neuroblastomas these genes carry H3K4me3 marks within their promoters. Thus, a failure of the epigenetic silencing of these genes during development may be an underlying factor responsible for neuroblastoma.

Niche required for inducing quiescent stem cells.

Quiescence is an important feature distinguishing stem cells (SCs) from other compartments for most SC systems. Evidence suggests that the quiescent state is directed by external cues expressed in the presumptive microenvironment, the niche, although the cellular and molecular nature of the niche remains obscure in most SC systems. Our group has been addressing this question using the melanocyte (MC) as a model, because MC SCs (MSCs) and other compartments are distinguished by their location in the hair follicle, the former in the bulge and the other in the hair matrix. On the basis of the gene expression profiles of MSCs, we developed a method to distinguish MSCs from other compartments by using their own characteristics. Using the new criterion for MSCs, we investigated the molecular cues that induce the quiescent MSCs from proliferating melanoblasts. Our study showed that fibroblast growth factor-2 (FGF-2), or an equivalent signal, is essential for inducing a set of MSC signatures, although additional signals required for inducing the ultimate MSCs remain to be identified.

Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction.

Interactions between the cell cycle machinery and transcription factors play a central role in coordinating terminal differentiation and proliferation arrest. We here show that cyclin-dependent kinase 6 (Cdk6) is specifically expressed in proliferating hematopoietic progenitor cells, and that Cdk6 inhibits transcriptional activation by Runx1, but not C/EBPalpha or PU.1. Cdk6 inhibits Runx1 activity by binding to the runt domain of Runx1, interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction. Cdk6 expression increased myeloid progenitor proliferation, and inhibited myeloid lineage-specific gene expression and terminal differentiation in vitro and in vivo. These effects of Cdk6 did not require Cdk6 kinase activity. Cdk6-mediated inhibition of granulocytic differentiation could be reversed by excess Runx1, consistent with Runx1 being the major target for Cdk6. We propose that Cdk6 downregulation in myeloid progenitors releases Runx1 from Cdk6 inhibition, thereby allowing terminal differentiation. Since Runx transcription factors play central roles in hematopoietic, neuronal and osteogenic lineages, this novel, noncanonical Cdk6 function may control terminal differentiation in multiple tissues and cell types.

What is a stem cell niche?

Niche has become the most important issue in stem cell biology, but it is still a hypothetical notion that cannot be defined in a better way than the microenvironment surrounding stem cells. Using a melanocyte stem cell system as a model, we have analyzed the cellular and molecular requirements for differentiation of quiescent stem cells. Our results demonstrate the multiple subsets within the stem cell compartment and thus suggests the complexity of niche.

Niche for normal and cancer stem cells.

An important issue in cancer therapy is the presence of a population that is resistant to anticancer treatment. This resistance has been partly ascribed to the presence of quiescent stem cells in a cancer population. However, how the quiescent state is induced in a proliferating cancer population is totally obscure. We think that our study on the stem cell system of pigment cells will provide some insight into the molecular basis for cancer stem cells, because the quiescent melanocyte stem cell would be the ideal model for understanding the process generating quiescent stem cells. In this article, we review our latest understanding of the quiescent stem cells of the melanocyte lineage by referring to some related topics of cancer stem cells.

Melanocyte system for studying stem cell niche.

There are many notions in stem cell biology that lack proof. The stem cell niche is the most typical example. While it is a convenient terminology for designating anything that supports stem cells, the cellular basis of the niche is poorly understood for many stem cell systems. In this chapter, we describe how useful the melanocyte system would be for investigating the molecular and cellular basis of the niche.

A complex linkage in the developmental pathway of endothelial and hematopoietic cells.

During normal vertebrate development, hematopoietic and endothelial cells form closely situated and interacting populations. Although the close proximity of cells to each other does not necessarily mean that they are relatives, accumulating evidence indicates that hematopoietic and endothelial cells are indeed close kin; they share common progenitors and each is able to become the other under certain circumstances. This article summarizes recent advances in the developmental relationship between hematopoietic and endothelial cells.

Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells.

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.

Functional blockade of platelet-derived growth factor receptor-beta but not of receptor-alpha prevents vascular smooth muscle cell accumulation in fibrous cap lesions in apolipoprotein E-deficient mice.

BACKGROUND: The vascular smooth muscle cell (VSMC) is the central cell component involved in the fibroproliferative response in atherogenesis. As the lesion advances, VSMCs migrate from the media into the subendothelial space, thereby forming fibrous plaque lesions. Platelet-derived growth factor (PDGF) has been known to be a potent chemoattractant and mitogen for SMCs, but the pathophysiological role of the 2 PDGF receptors, receptor-alpha (PDGFR-alpha) and receptor-beta (PDGFR-beta) in atherogenesis is poorly understood. To clarify this problem, we prepared antagonistic rat monoclonal antibodies, APA5 and APB5, against murine PDGFR-alpha and PDGFR-beta, respectively. METHODS AND RESULTS: Apolipoprotein E-deficient mice were fed a high-fat diet containing 0.3% cholesterol from 6 weeks of age and subjected to injection with 1 mg/d IP of either antibody from 12 to 18 weeks every other day. In the mice injected with APB5, the aortic atherosclerotic lesion size and the number of intimal VSMCs were reduced by 67% and 80%, respectively, compared with the control mice injected with irrelevant rat IgG. In contrast, the mice that received APA5 showed only minimal reduction of lesion size, and a large number of VSMCs were observed in the intima. In the intima of advanced lesions, APB5 immunolabeled VSMCs, whereas APA5 could detect VSMCs mainly in the media. CONCLUSIONS: These results indicate that PDGFR-beta plays a significant role in formation of fibrous atherosclerotic lesions and that regulation of the signal transduction through PDGFR-beta could affect atherogenesis in mice.

Origin of hematopoietic progenitors during embryogenesis.

It has been widely accepted that hematopoietic and endothelial cell lineages diverge from a common progenitor referred to as the hemangioblast. Recently, analyses of the potential of progenitor cells purified from mouse embryos as well as embryonic stem cells differentiating in vitro resolved intermediate stages between mesodermal cells and committed precursors for hematopoietic and endothelial cell lineages. There are two distinct hematopoietic cell lineages which have different origins, i.e., primitive hematopoietic lineage derived from mesoderm or hemangioblasts and definitive hematopoietic lineage derived from endothelial cells. The endothelium is suggested to provide a milieu in which the definitive hematopoietic lineage acquires multiple potentials.

Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

Molecular basis for hematopoietic/mesenchymal interaction during initiation of Peyer's patch organogenesis.

Mice deficient in lymphotoxin beta receptor (LTbetaR) or interleukin 7 receptor alpha (IL-7Ralpha) lack Peyer's patches (PPs). Deficiency in CXC chemokine receptor 5 (CXCR5) also severely affects the development of PPs. A molecular network involving these three signaling pathways has been implicated in PP organogenesis, but it remains unclear how they are connected during this process. We have shown that PP organogenesis is initiated at sites containing IL-7Ralpha(+) lymphoid cells and vascular cell adhesion molecule (VCAM)-1/intercellular adhesion molecule (ICAM)-1 expressing nonlymphoid elements. Here we characterize these lymphoid and nonlymphoid components in terms of chemokine signals. The lymphoid population expresses CXCR5 and has a strong chemotactic response to B lymphocyte chemoattractant (BLC). Importantly, chemokines produced by VCAM-1(+)ICAM-1(+) nonlymphoid cells mediate the recruitment of lymphoid cells. Furthermore, we show that these VCAM-1(+)ICAM-1(+) cells are mesenchymal cells that are activated by lymphoid cells through the LTbetaR to express adhesion molecules and chemokines. Thus, promotion of PP development relies on mutual interaction between mesenchymal and lymphoid cells.

Review: melanocyte migration and survival controlled by SCF/c-kit expression.

Melanocytes are derived from neural crest and migrate along the dorsolateral pathway to colonize the final destination in the skin. Stem cell factor and its receptor c-kit were identified as gene products of Sl and W mutant loci; both of them were known to have defects in melanocytes survival. In this review, we focus on the function of stem cell factor and c-kit in melanocyte migration and survival, which has become clearer in the last decade. By analysis of both molecules in wild-type and white spotting mutant mice, ligand and receptor set were shown to play multiple roles in the development of melanocytes in mouse ontogeny. Functional blockade of c-kit by specific monoclonal antibody illustrated distinct c-kit dependent and independent stages in melanocyte development. Finally, SCF transgene expression demonstrated that part of the c-kit dependent step is regulated by spatiotemporally specific ligand expression and also indicated the presence of c-kit independent melanocyte stem cells in postnatal skin.

Study on PDGF receptor beta pathway in glomerular formation in neonate mice.

The assembly of vascular endothelial cells (ECs) and smooth muscle cells is a critical event in the development of the cardiovascular system. Although the role of ECs in this event has been studied intensively, the cross-talk between the two cell components remains poorly understood. In this study, we blocked platelet-derived growth factor receptor (PDGFR) pathways in mice by antagonistic rat monoclonal antibody APB5 against murine PDGFR-beta and examined glomerular capillary formation.

Cell biology of vascular endothelial cells.

The number of molecules identified as being involved in the development of the vascular system has been increasing recently. Among those are secretory molecules and their receptors that collaborate in concert to regulate the process. Of note, however, is that we know little about the response of normal endothelial cells to different stimuli. In this study, we established a method of producing a normal endothelial population from embryonic stem cells. This new culture system was used to analyze the behavior of endothelial cells to various angiogenic stimuli. Our study demonstrated clearly that this culture system is extremely useful in revealing the behavior of developing endothelial cells. Implications derived from our observations are discussed.

Lactation defect in mice lacking the helix-loop-helix inhibitor Id2.

Id proteins are thought to be negative regulators of cell differentiation and positive regulators of cell proliferation. Mammary glands of Id2(-/-) female mice reveal severely impaired lobulo-alveolar development during pregnancy. Id2(-/-) mammary epithelia show no precocious maturation, but instead exhibit intrinsic defects in both cell proliferation and cell survival, implying that the role of Id2 in pregnant mammary epithelia is mainly stimulation of cell proliferation and support of cell viability. Expression studies of genes required for mammary gland development suggest Id2 to be a downstream or parallel factor of these genes. A decrease in the DNA binding activity of Stat5 was also observed in Id2(-/-) mammary glands at 7 days post-coitus. Our results indicate an indispensable role of Id2 in pregnant mammary glands.

Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.

We have identified a stromal cell-derived inducing activity (SDIA) that promotes neural differentiation of mouse ES cells. SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies. BMP4, which acts as an antineuralizing morphogen in Xenopus, suppresses SDIA-induced neuralization and promotes epidermal differentiation. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained from SDIA-treated ES cells. When transplanted, SDIA-induced dopaminergic neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase expression. Neural induction by SDIA provides a new powerful tool for both basic neuroscience research and therapeutic applications.

All B cells are progeny of endothelial cells: a new perspective.

We present here a speculative view of embryonic hematopoiesis. We do this with the hope of finding directions for future study, keeping in mind that our model may diverge from the real situation. However, we want to emphasize that previous models have neglected the possibility that endothelial cells (EC) represent a progenitor of hematopoietic cells (HPC). Emerging evidence, including our own, and previous histological studies argue for the presence of hemogenic EC. We discuss seemingly contradictory points of view of embryonic hematopoiesis, such as the origin of lymphogenic progenitors, and have shown that they may be resolved more satisfactorily by introducing this notion. Obviously, a good model should be a testable one, so we are currently developing experimental systems to demonstrate that all B cells are, indeed, the progeny of EC.

Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis.

Vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis. VEGF-C, however, is thought to stimulate the growth of lymphatic vessels because an expression of its specific receptor, VEGF receptor-3 (VEGFR-3), was demonstrated to be restricted to lymphatic vessels. Here we demonstrate that the inactivation of VEGFR-3 by a novel blocking monoclonal antibody (mAb) suppresses tumor growth by inhibiting the neo-angiogenesis of tumor-bearing tissues. Although VEGFR-3 is not expressed in adult blood vessels, it is induced in vascular endothelial cells of the tumor-bearing tissues. Hence, VEGFR-3 is another receptor tyrosine kinase involved in tumor-induced angiogenesis. Micro-hemorrhage in the tumor-bearing tissue was the most conspicuous histologic finding specific to AFL4 mAb-treated mice. Scanning microscopy demonstrated disruptions of the endothelial lining of the postcapillary venule, probably the cause of micro-hemorrhage and the subsequent collapse of the proximal vessels. These findings suggest the involvement of VEGFR-3 in maintaining the integrity of the endothelial lining during angiogenesis. Moreover, our results suggest that the VEGF-C/VEGFR-3 pathway may serve another candidate target for cancer therapy. (Blood. 2000;96:546-553)