Biomedical subjects

Timm Schroeder

Publications and source records attributed to Timm Schroeder.

11 recordsLinked to original sources

PubMed2006

MAPK phosphatase-1 represents a novel anti-inflammatory target of glucocorticoids in the human endothelium.

Glucocorticoids are well-established anti-inflammatory drugs thought to mainly act by inhibition of proinflammatory transcription factors like NF-kappaB. In recent years, however, transcription factor-independent mechanisms of glucocorticoid action have been proposed, namely the influence on MAPK pathways. Here we identify MAPK phosphatase-1 (MKP-1) as a pivotal mediator of the anti-inflammatory action of glucocorticoids in the human endothelium. We applied dexamethasone (Dex) to TNF-alpha-activated human endothelial cells and used the adhesion molecule E-selectin as inflammatory read-out parameter. Dex is known to reduce the expression of E-selectin, which is largely regulated by NF-kappaB. Here, we communicate that Dex at low concentrations (1-100 nM) markedly attenuates E-selectin expression without affecting NF-kappaB. Importantly, Dex is able to increase the expression of MKP-1, which causes an inactivation of TNF-alpha-induced p38 MAPK and mediates inhibition of E-selectin expression. In endothelial MKP-1(-/-) cells differentiated from MKP-1(-/-) embryonic stem cells and in MKP-1-silenced human endothelial cells, Dex did not inhibit TNF-alpha-evoked E-selectin expression. Thus, our findings introduce MKP-1 as a novel and crucial mediator of the anti-inflammatory action of glucocorticoids at low concentrations in the human endothelium and highlight MKP-1 as an important and promising anti-inflammatory drug target.

Animals↗

PubMed2006

Adrenomedullin/cyclic AMP pathway induces Notch activation and differentiation of arterial endothelial cells from vascular progenitors.

OBJECTIVE: The acquisition of arterial or venous identity is highlighted in vascular development. Previously, we have reported an embryonic stem (ES) cell differentiation system that exhibits early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors. In this study, we constructively induced differentiation of arterial and venous endothelial cells (ECs) in vitro to elucidate molecular mechanisms of arterial-venous specification. METHODS AND RESULTS: ECs were induced from VEGFR2+ progenitor cells with various conditions. VEGF was essential to induce ECs. Addition of 8bromo-cAMP or adrenomedullin (AM), an endogenous ligand-elevating cAMP, enhanced VEGF-induced EC differentiation. Whereas VEGF alone mainly induced venous ECs, 8bromo-cAMP (or AM) with VEGF supported substantial induction of arterial ECs. Stimulation of cAMP pathway induced Notch signal activation in ECs. The arterializing effect of VEGF and cAMP was abolished in recombination recognition sequence binding protein at the Jkappa site deficient ES cells lacking Notch signal activation or in ES cells treated with gamma-secretase inhibitor. Nevertheless, forced Notch activation by the constitutively active Notch1 alone did not induce arterial ECs. CONCLUSIONS: Adrenomedullin/cAMP is a novel signaling pathway to activate Notch signaling in differentiating ECs. Coordinated signaling of VEGF, Notch, and cAMP is required to induce arterial ECs from vascular progenitors.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2006

Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development.

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.

Cell Differentiation↗

PubMed2005

Common themes and cell type specific variations of higher order chromatin arrangements in the mouse.

BACKGROUND: Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes) with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. RESULTS: All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. CONCLUSION: Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation.

Animals↗

PubMed2005

Cdc42 is not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells.

Cdc42 is a small GTPase involved in the regulation of the cytoskeleton and cell polarity. To test whether Cdc42 has an essential role in the formation of filopodia or directed cell migration, we generated Cdc42-deficient fibroblastoid cells by conditional gene inactivation. We report here that loss of Cdc42 did not affect filopodium or lamellipodium formation and had no significant influence on the speed of directed migration nor on mitosis. Cdc42-deficient cells displayed a more elongated cell shape and had a reduced area. Furthermore, directionality during migration and reorientation of the Golgi apparatus into the direction of migration was decreased. However, expression of dominant negative Cdc42 in Cdc42-null cells resulted in strongly reduced directed migration, severely reduced single cell directionality, and complete loss of Golgi polarization and of directionality of protrusion formation toward the wound, as well as membrane blebbing. Thus, our data show that besides Cdc42 additional GTPases of the Rho-family, which share GEFs with Cdc42, are involved in the establishment and maintenance of cell polarity during directed migration.

Cell Adhesion↗

PubMed2005

Tracking hematopoiesis at the single cell level.

Despite intensive research, many longstanding questions of experimental hematology remain unsolved. One major reason is the fact that hematopoiesis is usually followed by analyzing populations of cells rather than individual cells, at few points in time during an experiment and without knowing (or quickly loosing) the cells' individual identities. The static picture yielded by this approach makes it impossible to appreciate the dynamic developmental processes leading to the generation of the full hematopoietic system from individual hematopoietic stem cells (HSCs). Real-time tracking of individual cells in culture, tissues, or whole organisms would be an extremely powerful approach to fully understand the developmental complexity of hematopoiesis. To this end, a computer-aided culture and bioimaging system is being developed to follow the fate of individual cells over long periods of time. This system is used to follow the development of multilineage cobblestone colonies from adult HSCs in stroma cocultures at the single cell level over many generations. To facilitate noninvasive detection of lineage commitment in these cultures, new subcellular forms of optimized fluorescent proteins have been developed to allow simultaneous marking of multiple hematopoietic lineages within the same animal.

Adult↗

PubMed2004

Generation of optimized yellow and red fluorescent proteins with distinct subcellular localization.

Fluorescent proteins (FPs) have revolutionized many aspects of cell biology and have become indispensable research tools. Today's increasingly complex experiments aiming to understand biological systems strongly depend on the availability of combinations of multiple FPs, which allow their distinguishable simultaneous detection in the same cell or tissue. Recently, the VENUS and DsRed. T4 FPs were described as the latest generation of yellow and red FPs. To increase the combinatorial possibilities when using these optimized FPs, we have generated and successfully tested seven new forms of VENUS and DsRed. T4 proteins with distinct subcellular localization. To facilitate their use as markers in biological experiments, bicistronic expression constructs, which have been optimized for robust expression in almost all mammalian developmental stages and cell types, were produced for the new FPs. In addition, several plasmids were created, which contain all necessary elements for inserting the reading frames of these FPs into specific gene loci in knock-in experiments without disrupting the reading frame of the endogenous gene.

Cell Line↗

PubMed2003

Notch signaling induces multilineage myeloid differentiation and up-regulates PU.1 expression.

Hemopoietic commitment is initiated by and depends on activation of transcription factors. However, it is unclear whether activation of lineage-affiliated transcription factors is extrinsically regulated by to date unknown agents or is the result of a cell autonomous program. Here we show that signaling by the Notch1 transmembrane receptor instructively induces myeloid differentiation of multipotent hemopoietic progenitor cells and concomitantly up-regulates the expression of the transcription factor PU.1. Transient activation of Notch1 signaling is sufficient to irreversibly reduce self-renewal of multipotent progenitor cells accompanied by increased and accelerated differentiation along the granulocyte, macrophage, and dendritic cell lineages. Activated Notch1 has no direct influence on apoptosis of multipotent progenitor cells, shows a weak inhibition of proliferation, and does not substitute for survival and proliferation signals provided by cytokines. Activated Notch1 directly increases PU.1 RNA levels, leading to a high concentration of PU.1 protein, which has been shown to direct myeloid differentiation. These findings identify Notch as an extrinsic regulator of myeloid commitment, and the lineage-affiliated transcription factor PU.1 as a specific direct target gene of Notch.

Animals↗

PubMed2003

Recombination signal sequence-binding protein Jkappa alters mesodermal cell fate decisions by suppressing cardiomyogenesis.

The transcription factor recombination signal sequence-binding protein Jkappa (RBP-J) is a key downstream element in the signaling pathway of all four mammalian Notch receptors that are critically involved in the control of embryonic and adult development. RBP-J-deficient mice display complex defects and die around day 9.5 postcoitum. Here, we investigate the function of RBP-J in the development of mesodermal cell lineages by using the OP9 stroma coculture system. RBP-J-deficient embryonic stem (ES) cells gave rise to cardiomyocytes, endothelial cells, and primitive and definitive hematopoietic cells. Thus, RBP-J-mediated signals are not required for generation of these cell types. However, when compared with parental RBP-J-expressing ES cells, cardiomyogenesis derived from RBP-J-deficient ES cells was increased. Repression over the cardiogenic pathway was restored by expressing RBP-J in RBP-J-deficient ES cells. Our data indicate that Notch signaling via RBP-J plays an important role for the correct specification of myocardial cell fates.

Animals↗

PubMed2003

Dendritic cells under influence of mouse cytomegalovirus have a physiologic dual role: to initiate and to restrict T cell activation.

The aim of this study is to analyze the dynamics of the mouse cytomegalovirus (MCMV)-dendritic cell (DC) interaction. Immature and mature DCs derived from the mouse stem cell line factor-dependent cell Paterson mixed potential were infected with a recombinant MCMV expressing green fluorescent protein. Infection of immature DCs resulted in DC activation and virus production, both of which may contribute to viral dissemination. The infection of mature DCs was nonproductive and was restricted to immediate-early and early viral protein expression. During early stages of MCMV infection, mature DCs up-regulated major histocompatibility complex (MHC) and costimulatory molecules and activated autologous, but not allogeneic, naive T cells. At later times of MCMV infection, DCs prevented T cell activation by down-regulation of MHC and costimulatory molecules. Thus, DCs under the influence of MCMV have a physiologic dual role: to initiate and to restrict T cell activation. The lack of immunostimulation in allogeneic settings may explain the increased risk of MCMV morbidity after allogeneic transplantation.

Animals↗

PubMed2003

Transient expression of PU.1 commits multipotent progenitors to a myeloid fate whereas continued expression favors macrophage over granulocyte differentiation.

OBJECTIVES: The Ets-family transcription factor PU.1 is expressed specifically in the hematopoietic system, in which it is absolutely required for the generation of B lymphocytes and macrophages. In contrast, overexpression of PU.1 blocks terminal differentiation of the erythroid lineage, in which it can act as an oncogene. In this study we used a multipotential progenitor cell line to examine the effects of PU.1 overexpression on myeloerythroid commitment within a single model system. MATERIALS AND METHODS: PU.1 cDNA was introduced transiently and stably into the multipotent, nonleukemic hemopoietic cell line FDCPmix. Transiently transfected cells were isolated by fluorescence-activated cell sorting within 18 hours of transfection. Stable transfectants were selected by antibiotic resistance over a number of weeks. The effects of short- and long-term overexpression of PU.1 on self-renewal, proliferation, and differentiation were investigated. RESULTS: A transient pulse of expression in multipotent progenitor cells eliminated the options of self-renewal and erythroid differentiation, resulting in commitment to the myeloid lineage. However, this transient pulse of expression did not affect the subsequent lineage choice of bipotent granulocyte/macrophage progenitors. In contrast, continuous expression of PU.1 resulted in a strong bias toward macrophage rather than granulocyte differentiation. CONCLUSIONS: These results demonstrate promyeloid effects of PU.1 at two distinct stages of hematopoiesis.

Animals↗