技术资料

Dendritic cell (DC) differentiation is regulated by stroma via a network of soluble and cell-bound factors. Notch is one of the major elements of this network. Its role in DC differentiation,however,is controversial. Here,we demonstrate that activation of Notch signaling in hematopoietic progenitor cells (HPCs) promoted differentiation of conventional DCs via activation of the canonical Wingless (Wnt) pathway. Inhibition of the Wnt pathway abrogated the effect of Notch on DC differentiation. The fact that activation of the Wnt pathway in Notch-1-deficient embryonic stem cells restored DC differentiation indicates that Wnt signaling is downstream of the Notch pathway in regulating DC differentiation. Notch signaling activated the Wnt pathway in HPCs via expression of multiple members of the Frizzled family of Wnt receptors,which was directly regulated by the CSL (RPB-Jkappa) transcription factor. Thus,these data suggest a model of DC differentiation via cooperation between Wnt and Notch pathways. View Publication

DNA methylation,histone modifications,and nucleosomal occupancy collaborate to cause silencing of tumor-related genes in cancer. The development of drugs that target these processes is therefore important for cancer therapy. Inhibitors of DNA methylation and histone deacetylation have been approved by the Food and Drug Administration for treatment of hematologic malignancies. However,drugs that target other mechanisms still need to be developed. Recently,3-deazaneplanocin A (DZNep) was reported to selectively inhibit trimethylation of lysine 27 on histone H3 (H3K27me3) and lysine 20 on histone H4 (H4K20me3) as well as reactivate silenced genes in cancer cells. This finding opens the door to the pharmacologic inhibition of histone methylation. We therefore wanted to further study the mechanism of action of DZNep in cancer cells. Western blot analysis shows that DZNep globally inhibits histone methylation and is not selective. Two other drugs,sinefungin and adenosine dialdehyde,have similar effects as DZNep on H3K27me3. Intriguingly,chromatin immunoprecipitation of various histone modifications and microarray analysis show that DZNep acts through a different pathway than 5-aza-2'-deoxycytidine,a DNA methyltransferase inhibitor. These observations give us interesting insight into how chromatin structure affects gene expression. We also determined the kinetics of gene activation to understand if the induced changes were somatically heritable. We found that upon removal of DZNep,gene expression is reduced to its original state. This suggests that there is a homeostatic mechanism that returns the histone modifications to their ground state" after DZNep treatment. Our data show the strong need for further development of histone methylation inhibitors." View Publication

The understanding of the function of alpha(1)-adrenergic receptors in the brain has been limited due to a lack of specific ligands and antibodies. We circumvented this problem by using transgenic mice engineered to overexpress either wild-type receptor tagged with enhanced green fluorescent protein or constitutively active mutant alpha(1)-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing alpha(1A)-adrenergic receptor subtype-expressing cells with a migratory morphology in the adult subventricular zone that coexpressed markers of neural stem cell and/or progenitors. Incorporation of 5-bromo-2-deoxyuridine in vivo increased in neurogenic areas in adult alpha(1A)-adrenergic receptor transgenic mice or normal mice given the alpha(1A)-adrenergic receptor-selective agonist,cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of alpha(1)-adrenergic receptor subtypes,and stimulation of these receptors resulted in increased expression of the alpha(1B)-adrenergic receptor subtype,proneural basic helix-loop-helix transcription factors,and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. alpha(1)-Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However,in adult normal neurospheres,alpha(1)-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo,S100-positive glial and betaIII tubulin neuronal progenitors colocalized with either alpha(1)-adrenergic receptor subtype in the olfactory bulb. Our results indicate that alpha(1)-adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases. View Publication

Objective: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) constitute unique sources of pluripotent cells,although the molecular mechanisms involved in their differentiation into specific lineages are just beginning to be defined. Here we evaluated the ability of MEDII (medium conditioned by HepG2 cells,a human hepatocarcinoma cell line) to selectively enhance generation of mesodermal derivatives,including hematopoietic cells,from hESCs and hiPSCs. Materials and Methods: Test cells were exposed to MEDII prior to being placed in conditions that promote embryoid body (EB) formation. Hematopoietic activity was measured by clonogenic assays,flow cytometry,quantitative real-time polymerase chain reaction of specific transcript complementary DNAs and the ability of cells to repopulate sublethally irradiated nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice for almost 1 year. Results: Exposure of both hESCs and hiPSCs to MEDII induced a rapid and preferential differentiation of hESCs into mesodermal elements. Subsequently produced EBs showed a further enhanced expression of transcripts characteristic of multiple mesodermal lineages,and a concurrent decrease in endodermal and ectodermal cell transcripts. Frequency of all types of clonogenic hematopoietic progenitors in subsequently derived EBs was also increased. In vivo assays of MEDII-treated hESC-derived EBs also showed they contained cells able to undertake low-level but longterm multilineage repopulation of primary and secondary nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice. Conclusions: MEDII treatment of hESCs and hiPSCs alike selectively enhances their differentiation into mesodermal cells and allows subsequent generation of detectable levels of hematopoietic progenitors with in vitro and in vivo differentiating activity. ?? 2009 ISEH - Society for Hematology and Stem Cells. View Publication

The control of tyrosine phosphorylation depends on the fine balance between kinase and phosphatase activities. Protein tyrosine phosphatase 1B (PTP-1B) and T cell protein tyrosine phosphatase (TC-PTP) are 2 closely related phosphatases known to control cytokine signaling. We studied the functional redundancy of PTP-1B and TC-PTP by deleting 1 or both copies of these genes by interbreeding TC-PTP and PTP-1B parental lines. Our results indicate that the double mutant (tcptp(-/-)ptp1b(-/-)) is lethal at day E9.5-10.5 of embryonic development with constitutive phosphorylation of Stat1. Mice heterozygous for TC-PTP on a PTP-1B-deficient background (tcptp(+/-)ptp1b(-/-)) developed signs of inflammation. Macrophages from these animals were highly sensitive to IFN-gamma,as demonstrated by increased Stat1 phosphorylation and nitric oxide production. In addition,splenic T cells demonstrated increased IFN-gamma secretion capacity. Mice with deletions of single copies of TC-PTP and PTP-1B (tcptp(+/-)ptp1b(+/-)) exhibited normal development,confirming that these genes are not interchangeable. Together,these data indicate a nonredundant role for PTP-1B and TC-PTP in the regulation of IFN signaling. View Publication

After DNA replication,sister chromatids must be untangled,or decatenated,before mitosis so that chromatids do not tear during anaphase. Topoisomerase IIalpha (Topo IIalpha) is the major decatenating enzyme. Topo IIalpha inhibitors prevent decatenation,causing cells to arrest during mitosis. Here we report that acute myeloid leukemia cells fail to arrest at the mitotic decatenation checkpoint,and their progression through this checkpoint is regulated by the DNA repair component Metnase (also termed SETMAR). Metnase contains a SET histone methylase and transposase nuclease domain,and is a component of the nonhomologous end-joining DNA double-strand break repair pathway. Metnase interacts with Topo IIalpha and enhances its decatenation activity. Here we show that multiple types of acute leukemia cells have an attenuated mitotic arrest when decatenation is inhibited and that in an acute myeloid leukemia (AML) cell line this is mediated by Metnase. Of further importance,Metnase permits continued proliferation of these AML cells even in the presence of the clinical Topo IIalpha inhibitor VP-16. In vitro,purified Metnase prevents VP-16 inhibition of Topo IIalpha decatenation of tangled DNA. Thus,Metnase expression levels may predict AML resistance to Topo IIalpha inhibitors,and Metnase is a potential therapeutic target for small molecule interference. View Publication

Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells,but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule,kenpaullone,in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately,such compounds may provide mechanistic insight into the reprogramming process. View Publication

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. They can maintain an undifferentiated state indefinitely and can differentiate into derivatives of all three germ layers,namely ectoderm,endoderm and mesoderm. Although much progress has been made in the propagation and differentiation of ES cells,induction of photoreceptors has generally required coculture with or transplantation into developing retinal tissue. Here,we describe a protocol for generating retinal cells from ES cells by stepwise treatment with defined factors. This method preferentially induces photoreceptor and retinal pigment epithelium (RPE) cells from mouse and human ES cells. In our protocol,differentiation of RPE and photoreceptors from mouse ES cells requires 28 d and the differentiation of human ES cells into mature RPE and photoreceptors requires 120 and 150 d,respectively. This differentiation system and the resulting pluripotent stem cell-derived retinal cells will facilitate the development of transplantation therapies for retinal diseases,drug testing and in vitro disease modeling. It will also improve our understanding of the development of the central nervous system,especially the eye. View Publication

BACKGROUND Interleukin (IL-6) and transforming growth factor (TGF)-beta have been shown to play a role in skin development and maintenance. OBJECTIVES A link between these two cytokines has yet to be identified and therefore in this study we investigated the modulation of TGF-beta1 and TGF-beta type 2 receptor (TGF-betaR2) by IL-6 in skin. METHODS An IL-6 knockout (IL-6KO) fibroblast-populated lattice model and intradermal injections of IL-6 into unwounded IL-6KO mice were used to investigate the direct effects of IL-6 treatment on TGF-beta and TGF-betaR2 expression and to determine the signalling mechanism. In addition,IL-6KO and C57BL/6 control mice were wounded by a 4-mm punch biopsy to monitor expression of TGF-beta1 and TGF-betaR2 within a wound over time. The expression of TGF-beta1 and TGF-betaR2 was assessed by real-time quantitative polymerase chain reaction,enzyme-linked immunosorbent assay and immunohistology. RESULTS Recombinant IL-6 treatment of IL-6KO lattices and intradermal injections of IL-6 showed a significant induction of TGF-beta1 mRNA and protein,with TGF-beta1 expression localized in the dermis,while TGF-betaR2 expression was primarily in the epidermis in IL-6KO mice. During healing,the expression of TGF-beta1 and TGF-betaR2 mRNA was significantly greater in unwounded and 7-day-old wounds from wild-type mice; however,protein expression did not differ. Treatment with signal transduction inhibitors indicated that IL-6 modulates TGF-beta through a mitogen-activated protein kinase/extracellular signal-regulated kinase (Mapk/Erk)-dependent mechanism. CONCLUSION These studies indicate that IL-6 has the ability to modulate the expression of TGF-beta and TGF-betaR2 to varying degrees in the skin,which may provide a possible mechanism for defining the role of IL-6 in skin maintenance and a new association of IL-6 with TGF-beta in pathologies associated with fibrosis. View Publication

This study investigated the effect of mechanical load on human mesenchymal stem cell (hMSC) differentiation under different exogenous transforming growth factor-beta1 (TGF-beta(1)) concentrations (0,1 or 10 ng/ml).The role of the TGF-beta signalling pathway in this process was also studied. Human MSCs were seeded into fibrin-biodegradable polyurethane scaffolds at a cell density of 5 x 10(6) cells per scaffold and stimulated using our bioreactor. One hour of surface motion superimposed on cyclic compression was applied once a day over seven consecutive days. Scaffolds were analysed for gene expression,DNA content and glycosaminoglycan amount. Addition of TGF-beta(1) in the culture medium was sufficient to induce chondrogenesis of hMSCs. Depending on the TGF-beta(1) concentration of the culture medium,mechanical load stimulated chondrogenesis of hMSCs compared to the unloaded scaffolds,with a much stronger effect on gene expression at lower TGF-beta(1) concentrations. With TGF-beta(1) absent in the culture medium,mechanical load stimulated gene transcripts and protein synthesis of TGF-beta(1) and TGF-beta(3). TGF-beta type I receptor inhibitor LY364947 blocked the up-regulation on TGF-beta(1) and TGF-beta(3) production stimulated by mechanical load,and also blocked the chondrogenesis of hMSCs. Taken together,these findings suggest that mechanical load promotes chondrogenesis of hMSCs through TGF-beta pathway by up-regulating TGF-beta gene expression and protein synthesis. View Publication

Garcinol (camboginol) from the fruit rind of Guttiferae species shows anti-carcinogenic and anti-inflammatory properties,but the underlying molecular mechanisms are unclear. Here we show that garcinol potently interferes with 5-lipoxygenase (EC 7.13.11.34) and microsomal prostaglandin (PG)E2 synthase (mPGES)-1 (EC 5.3.99.3),enzymes that play pivotal roles in inflammation and tumorigenesis. In cell-free assays,garcinol inhibited the activity of purified 5-lipoxygenase and blocked the mPGES-1-mediated conversion of PGH2 to PGE2 with IC50 values of 0.1 and 0.3 microM,respectively. Garcinol suppressed 5-lipoxygenase product formation also in intact human neutrophils and reduced PGE2 formation in interleukin-1beta-stimulated A549 human lung carcinoma cells as well as in human whole blood stimulated by lipopolysaccharide. Moreover,garcinol interfered with isolated cyclooxygenase (COX)-1 (EC 1.14.99.1,IC50 = 12 microM) and with the formation of COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and thromboxane B2 in human platelets. In contrast,neither Ca2+-ionophore (A23187)-induced arachidonic acid release in neutrophils nor COX-2 activity in A549 cells or whole blood,measured as formation of 6-keto PGF1alpha,or isolated human recombinant COX-2 were significantly affected by garcinol (textless or = 30 microM). Together,the high potency of garcinol to selectively suppress PGE2 synthesis and 5-lipoxygenase product formation provides a molecular basis for the anti-inflammatory and anti-carcinogenic effects of garcinol and rationalizes its therapeutic use. View Publication

Hematopoiesis is dependent upon the bone marrow microenvironment,which is comprised of multiple mesenchymal cell types,including fibroblasts,endothelial cells,osteoblasts,and stroma progenitors. The canonical Wnt signaling pathway,which relies on the beta-catenin protein to mediate its signal,is necessary for the normal development of mesenchymal tissue. We hypothesized that canonical Wnt signaling regulates the cellular composition and function of the bone marrow microenvironment. We observed that a beta-catenin-deficient bone marrow microenvironment maintained hematopoietic stem cells but exhibited a decreased capacity to support primitive hematopoietic cells. These results correlated with decreased numbers of osteoblasts and with decreased production of basic fibroblast growth factor,stem cell factor,and vascular cell adhesion molecule-1. From these data,we propose a model in which beta-catenin in the microenvironment is required noncell autonomously for long-term maintenance of hematopoietic progenitors. View Publication