Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/ thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2 ؊/؊ embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2 ؊/؊ embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.Reactive oxygen species (ROS)-generated mainly as a byproduct of the respiratory chain or by oxidases-are implicated in the pathogenesis and pathophysiology of a variety of human diseases such as cancer, cardiovascular, and degenerative disorders. A variety of cellular antioxidant systems control the balance of free intra-and extracellular oxygen radicals. Previous efforts have addressed the physiological role of superoxide dismutases, catalases, and glutathione (GSH) peroxidases in vivo, but the role of the thioredoxin/thioredoxin reductase/ peroxiredoxin system in ROS removal has only recently attracted attention.Thioredoxins are small redox-active proteins with an essential function in DNA metabolism and repair, transcription, and cell-cell communication (1). Acting through peroxiredoxins, they also efficiently protect cells from oxidative damage (27). Cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins are required for proliferation and protection from apoptosis during early embryogenesis (26). Moreover, in chicken B cells, Trx2 is critically involved in the regulation of mitochondriondependent apoptosis (37). More recently, heart-specific overexpression of dominant-negative Trx1 was shown to be associated with increased oxidative stress and cardiac hypertrophy in mice (39).Trx activities are governed by thioredoxin reductases (TrxRs) that, in turn, use NADPH/H ϩ as the reducing agent (23). TrxRs are members of the pyridine nucleotide-disulfide oxidoreductase family, form homodimers, and possess two interacting redox-active centers. The C-terminal redox center contains a catalytically important selenocysteine (SeCys) (9,17,41). In mammals, three TrxRs...
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 Jκ site deficient ES cells lacking Notch signal activation or in ES cells treated with γ-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.
The expression of Notch receptors on hematopoietic cells and of cognate ligands on bone marrow stromal cells suggests a possible role for Notch signalling in the regulation of hematopoiesis. In order to assess the involvement of Notch1 signalling in myelopoiesis, 32D myeloid progenitor cell lines were engineered to permit the conditional induction of the constitutively active intracellular domain of murine Notch1 (mN1 IC ) by the 4-hydroxytamoxifen-inducible system. The induction of mN1 IC resulted in accelerated and increased granulocytic differentiation. These effects were observed under growth conditions that support differentiation and, to a lesser degree, under conditions that normally promote self-renewal. Transient transfection of mN1 IC deletion mutants showed that the differentiation promoting activity correlated with RBP-J transactivation. Furthermore, expression of a transcriptionally active derivative of RBP-J (RBP-J± VP16) increased myeloid differentiation. To test further the role of Notch signalling in a physiological context, 32D cells expressing mNotch1 were cultured on ®broblast layers that either expressed or did not express the Notch ligand Jagged1. Similar to the induction of mN1 IC , Jagged1 accelerated granulocytic differentiation of 32D cells. Taken together, our data suggest that activation of mNotch1 promotes myeloid differentiation via RBP-J transactivation.
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.
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