scholarly journals Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling

2007 ◽  
Vol 404 (2) ◽  
pp. 235-245 ◽  
Author(s):  
Christine A. Biondi ◽  
Debipriya Das ◽  
Michael Howell ◽  
Ayesha Islam ◽  
Elizabeth K. Bikoff ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Transforming Growth Factor ◽  
Es Cells ◽  
Nuclear Export Signal ◽  
Embryonic Stem ◽  
Nucleocytoplasmic Shuttling ◽  
Mouse Development ◽  
Developmental Defects

Smad4 in partnership with R-Smads (receptor-regulated Smads) activates TGF-β (transforming growth factor-β)-dependent signalling pathways essential for early mouse development. Smad4 null embryos die shortly after implantation due to severe defects in cell proliferation and visceral endoderm differentiation. In the basal state, Smad4 undergoes continuous shuttling between the cytoplasm and the nucleus due to the combined activities of an N-terminal NLS (nuclear localization signal) and an NES (nuclear export signal) located in its linker region. Cell culture experiments suggest that Smad4 nucleocytoplasmic shuttling plays an important role in TGF-β signalling. In the present study we have investigated the role of Smad4 shuttling in vivo using gene targeting to engineer two independent mutations designed to eliminate Smad4 nuclear export. As predicted this results in increased levels of Smad4 in the nucleus of homozygous ES cells (embryonic stem cells) and primary keratinocytes, in the presence or absence of ligand. Neither mutation affects Smad4 expression levels nor its ability to mediate transcriptional activation in homozygous cell lines. Remarkably mouse mutants lacking the Smad4 NES develop normally. Smad4 NES mutants carrying one copy of a Smad4 null allele also fail to display developmental defects. The present study clearly demonstrates that Smad4 nucleocytoplasmic shuttling is not required for embryonic development or tissue homoeostasis in normal, healthy adult mice.

scholarly journals Nucleocytoplasmic Shuttling Modulates Activity and Ubiquitination-Dependent Turnover of SUMO-Specific Protease 2

2006 ◽  
Vol 26 (12) ◽  
pp. 4675-4689 ◽  
Author(s):  
Yoko Itahana ◽  
Edward T. H. Yeh ◽  
Yanping Zhang
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Shuttling ◽  
Leptomycin B ◽  
Sumo Protease ◽  
Specific Protease ◽  
Localization Signal ◽  
Modified Proteins ◽  
Export Signal

ABSTRACT Small ubiquitin-related modifier (SUMO) proteins are conjugated to numerous polypeptides in cells, and attachment of SUMO plays important roles in regulating the activity, stability, and subcellular localization of modified proteins. SUMO modification of proteins is a dynamic and reversible process. A family of SUMO-specific proteases catalyzes the deconjugation of SUMO-modified proteins. Members of the Sentrin (also known as SUMO)-specific protease (SENP) family have been characterized with unique subcellular localizations. However, little is known about the functional significance of or the regulatory mechanism derived from the specific localizations of the SENPs. Here we identify a bipartite nuclear localization signal (NLS) and a CRM1-dependent nuclear export signal (NES) in the SUMO protease SENP2. Both the NLS and the NES are located in the nonhomologous domains of SENP2 and are not conserved among other members of the SENP family. Using a series of SENP2 mutants and a heterokaryon assay, we demonstrate that SENP2 shuttles between the nucleus and the cytoplasm and that the shuttling is blocked by mutations in the NES or by treating cells with leptomycin B. We show that SENP2 can be polyubiquitinated in vivo and degraded through proteolysis. Restricting SENP2 in the nucleus by mutations in the NES impairs its polyubiquitination, whereas a cytoplasm-localized SENP2 made by introducing mutations in the NLS can be efficiently polyubiquitinated, suggesting that SENP2 is ubiquitinated in the cytoplasm. Finally, treating cells with MG132 leads to accumulation of polyubiquitinated SENP2, indicating that SENP2 is degraded through the 26S proteolysis pathway. Thus, the function of SENP2 is regulated by both nucleocytoplasmic shuttling and polyubiquitin-mediated degradation.

scholarly journals Transforming growth factor (TGF)beta, fibroblast growth factor (FGF) and retinoid signalling pathways promote pancreatic exocrine gene expression in mouse embryonic stem cells

2004 ◽  
Vol 379 (3) ◽  
pp. 749-756 ◽  
Author(s):  
Anouchka SKOUDY ◽  
Meritxell ROVIRA ◽  
Pierre SAVATIER ◽  
Franz MARTIN ◽  
Trinidad LEÓN-QUINTO ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Signalling Pathways ◽  
Fibroblast Growth ◽  
Development And Differentiation

Extracellular signalling cues play a major role in the activation of differentiation programmes. Mouse embryonic stem (ES) cells are pluripotent and can differentiate into a wide variety of specialized cells. Recently, protocols designed to induce endocrine pancreatic differentiation in vitro have been designed but little information is currently available concerning the potential of ES cells to differentiate into acinar pancreatic cells. By using conditioned media of cultured foetal pancreatic rudiments, we demonstrate that ES cells can respond in vitro to signalling pathways involved in exocrine development and differentiation. In particular, modulation of the hedgehog, transforming growth factor β, retinoid, and fibroblast growth factor pathways in ES cell-derived embryoid bodies (EB) resulted in increased levels of transcripts encoding pancreatic transcription factors and cytodifferentiation markers, as demonstrated by RT-PCR. In EB undergoing spontaneous differentiation, expression of the majority of the acinar genes (i.e. amylase, carboxypeptidase A and elastase) was induced after the expression of endocrine genes, as occurs in vivo during development. These data indicate that ES cells can undergo exocrine pancreatic differentiation with a kinetic pattern of expression reminiscent of pancreas development in vivo and that ES cells can be coaxed to express an acinar phenotype by activation of signalling pathways known to play a role in pancreatic development and differentiation.

Gene-trap expression screening to identify endothelial-specific genes

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 711-718 ◽  
Author(s):  
Masanori Hirashima ◽  
Alan Bernstein ◽  
William L. Stanford ◽  
Janet Rossant
Keyword(s):  
Endothelial Cells ◽  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transforming Growth Factor Β ◽  
Gene Trap ◽  
Lacz Reporter ◽  
Vascular Differentiation ◽  
Expression Screening

AbstractThe endothelial cell is a key cellular component for blood vessel formation. Many signaling receptors expressed in endothelial cells play critical roles in vascular development during embryogenesis. However, downstream response genes required for vascular differentiation are still not clearly identified. Here we describe the development of a protocol for gene-trap expression screening in embryonic stem (ES) cells for endothelial-specific genes. ES cells were differentiated into endothelial cells on an OP9 feeder cell layer in 96-well plates. In a pilot screen, 5 gene-trapped ES cell lines showed an up-regulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was endoglin, an endothelial-specific transforming growth factor-β type III receptor, and another was ASPP1, a p53-binding protein. In vivo expression analysis of the lacZ reporter confirmed that both genes are specifically expressed in endothelial cells during early mouse embryogenesis. Gene-trap expression screening can thus be used to identify early endothelial-specific genes and analyze their function in mice.

scholarly journals Mice Deficient for the Ets Transcription Factor Elk-1 Show Normal Immune Responses and Mildly Impaired Neuronal Gene Activation

2004 ◽  
Vol 24 (1) ◽  
pp. 294-305 ◽  
Author(s):  
Francesca Cesari ◽  
Stephan Brecht ◽  
Kristina Vintersten ◽  
Lam Giang Vuong ◽  
Matthias Hofmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Embryonic Stem ◽  
Adult Life ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Mouse Development ◽  
Serum Response ◽  
Deficient Mice

ABSTRACT The transcription factor Elk-1 belongs to the ternary complex factor (TCF) subfamily of Ets proteins. TCFs interact with serum response factor to bind jointly to serum response elements in the promoters of immediate-early genes (IEGs). TCFs mediate the rapid transcriptional response of IEGs to various extracellular stimuli which activate mitogen-activated protein kinase signaling. To investigate physiological functions of Elk-1 in vivo, we generated Elk-1-deficient mice by homologous recombination in embryonic stem cells. These animals were found to be phenotypically indistinguishable from their wild-type littermates. Histological analysis of various tissues failed to reveal any differences between Elk-1 mutant and wild-type mice. Elk-1 deficiency caused no changes in the proteomic displays of brain or spleen extracts. Also, no immunological defects could be detected in mice lacking Elk-1, even upon infection with coxsackievirus B3. In mouse embryonic fibroblasts, Elk-1 was dispensable for c-fos and Egr-1 transcriptional activation upon stimulation with serum, lysophosphatidic acid, or tetradecanoyl phorbol acetate. However, in brains of Elk-1-deficient mice, cortical and hippocampal CA1 expression of c-fos, but not Egr-1 or c-Jun, was markedly reduced 4 h following kainate-induced seizures. This was not accompanied by altered patterns of neuronal apoptosis. Collectively, our data indicate that Elk-1 is essential neither for mouse development nor for adult life, suggesting compensatory activities by other TCFs.

scholarly journals Kinetic Analysis of Smad Nucleocytoplasmic Shuttling Reveals a Mechanism for Transforming Growth Factor β-Dependent Nuclear Accumulation of Smads

2005 ◽  
Vol 25 (22) ◽  
pp. 9845-9858 ◽  
Author(s):  
Bernhard Schmierer ◽  
Caroline S. Hill
Keyword(s):  
Green Fluorescent Protein ◽  
Growth Factor ◽  
Nuclear Export ◽  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Transforming Growth Factor Β ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Shuttling ◽  
Green Fluorescent

ABSTRACT Upon transforming growth factor β (TGF-β) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. We find that TGF-β-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-β-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-β-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties.

scholarly journals Nuclear Export of the Transcription Factor NirA Is a Regulatory Checkpoint for Nitrate Induction in Aspergillus nidulans

2006 ◽  
Vol 27 (3) ◽  
pp. 791-802 ◽  
Author(s):  
Andreas Bernreiter ◽  
Ana Ramon ◽  
Javier Fernández-Martínez ◽  
Harald Berger ◽  
Lidia Araújo-Bazan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Nidulans ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Constitutive Expression ◽  
Nuclear Accumulation ◽  
Nuclear Retention ◽  
Nitrate Induction

ABSTRACT NirA, the specific transcription factor of the nitrate assimilation pathway of Aspergillus nidulans, accumulates in the nucleus upon induction by nitrate. NirA interacts with the nuclear export factor KapK, which bridges an interaction with a protein of the nucleoporin-like family (NplA). Nitrate induction disrupts the NirA-KapK interaction in vivo, whereas KapK associates with NirA when this protein is exported from the nucleus. A KpaK leptomycin-sensitive mutation leads to inducer-independent NirA nuclear accumulation in the presence of the drug. However, this does not lead to constitutive expression of the genes controlled by NirA. A nirA c 1 mutation leads to constitutive nuclear localization and activity, remodeling of chromatin, and in vivo binding to a NirA upstream activation sequence. The nirA c 1 mutation maps in the nuclear export signal (NES) of the NirA protein. The NirA-KapK interaction is nearly abolished in NirAc1 and NirA proteins mutated in canonical leucine residues in the NirA NES. The latter do not result in constitutively active NirA protein, which implies that nuclear retention is necessary but not sufficient for NirA activity. The results are consistent with a model in which activation of NirA by nitrate disrupts the interaction of NirA with the NplA/KapK nuclear export complex, thus resulting in nuclear retention, leading to AreA-facilitated DNA binding of the NirA protein and subsequent chromatin remodeling and transcriptional activation.

ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1343-1351
Author(s):  
A.M. Muller ◽  
E.A. Dzierzak
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Hematopoietic Stem ◽  
Developmental Potential

While hematopoietic stem cells from adult and fetal stages of murine development are capable of long term reconstitution of all mature blood lineages in vivo, embryonic hematopoietic stem cell repopulation in vivo has proved difficult. It is thought that there are many fewer hematopoietic stem cells in the embryo than in the fetal/adult stages of mouse development and that these cells possess a different developmental potential. One source of such cells are embryonic stem (ES) cells which can differentiate into most mature blood lineages in vitro. We have therefore used transplantation of differentiated ES cells to assess the hematopoietic potential of embryonic hematopoietic cells in vivo. We demonstrate here that precursors obtained from in vitro cultures of normal ES cells can contribute only to restricted and limited hematopoiesis in a mouse without leading to tumour formation. Repopulation occurs for greater than 6.5 months at levels ranging from 0.1% to 6% in B and T cell lineages in peripheral blood. In contrast to in vitro colony data demonstrating the myeloid lineage developmental potential of ES cells, no donor-derived myeloid repopulation was observed in CFU-S assays and no macrophage and mast cells were found in long term repopulated recipients. Thus, the hematopoietic potential of ES cells in vivo is limited to low levels of repopulation and is restricted to the lymphoid lineage.

scholarly journals TGF-β2 treatment enhances cytoprotective factors released from embryonic stem cells and inhibits apoptosis in infarcted myocardium

2011 ◽  
Vol 300 (4) ◽  
pp. H1442-H1450 ◽  
Author(s):  
Dinender K. Singla ◽  
Reetu D. Singla ◽  
Stephanie Lamm ◽  
Carley Glass
Keyword(s):  
Cell Culture ◽  
Cardiac Function ◽  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Tunel Staining ◽  
Beneficial Effects ◽  
Infarcted Heart ◽  
Induced Apoptosis

We investigated whether factors released from mouse embryonic stem (ES) cells primed with and without transforming growth factor (TGF)-β2 inhibit iodoacetic acid (IAA)- and H2O2-induced apoptosis in the cell culture system as well as after transplantation in the infarcted heart. We generated conditioned media (CMs) from ES cells primed with and without TGF-β2 and determined their effects on IAA- and H2O2-induced apoptosis in H9c2 cells. We also transplanted both ES-CMs in the infarcted heart to determine the effects on apoptosis and cardiac function after myocardial infarction (MI) at day (D)1 and D14. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, apoptotic ELISA, and cell viability data demonstrated significantly ( P < 0.05) reduced apoptosis with ES-CM compared with controls in both cell culture models. Moreover, TGF-β2-primed ES-CM (T-ES-CM) demonstrated enhanced beneficial effects, with further reduced ( P < 0.05) apoptosis compared with ES-CM, suggesting the a presence of additional cytoprotective released factors after TGF-β2 treatment. Next, our in vivo apoptosis data suggested significant decrease in apoptosis with both ES-CMs compared with MI alone at D1 and D14. Notably, T-ES-CM demonstrated significant ( P < 0.05) inhibition of apoptosis and fibrosis with improved cardiac function compared with ES-CM at D14, whereas no such effects were observed at D1. Next, we confirmed that apoptosis is mediated through a prosurvival Akt pathway. Moreover, we determined that after TGF-β2 treatment there was a two- to fivefold increase in cytoprotective released factors (interleukin-10, stem cell factor, tissue inhibitor of matrix metalloproteinase-1, and VEGF) with T-ES-CM compared with ES-CM. In conclusion, we suggest that factors released from ES cells with and without TGF-β2 treatment contain antiapoptotic factors that inhibit apoptosis in vitro and in vivo. We also suggest that T-ES-CM demonstrates additional beneficial effects that provide useful information for future therapeutic applications in regenerative medicine.

scholarly journals Transforming Growth Factor β-Independent Shuttling of Smad4 between the Cytoplasm and Nucleus

2000 ◽  
Vol 20 (23) ◽  
pp. 9041-9054 ◽  
Author(s):  
Christophe E. Pierreux ◽  
Francisco J. Nicolás ◽  
Caroline S. Hill
Keyword(s):  
Growth Factor ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Transforming Growth Factor ◽  
Nuclear Export Signal ◽  
Transforming Growth Factor Β ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Signaling Complex ◽  
Localization Signal

ABSTRACT Smad4 plays a pivotal role in all transforming growth factor β (TGF-β) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-β signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-β signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-β signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

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