scholarly journals A Key Cytoskeletal Regulator of Ubiquitination Amplifies TGFβ Signaling During Mouse Developmental Vascular Patterning

2016 ◽  
Author(s):  
Ronak Shetty ◽  
Divyesh Joshi ◽  
Mamta Jain ◽  
Madavan Vasudevan ◽  
Jasper Chrysolite Paul ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
De Novo ◽  
Yolk Sac ◽  
Dual Function ◽  
Tgfβ Signaling ◽  
Vascular Patterning ◽  
Cytoskeletal Remodeling ◽  
Tumour Metastasis ◽  
Microtubule Stability ◽  
Tgfβ Receptor

AbstractVascular development involves de novo formation of a capillary plexus, which is then pruned and remodeled by angiogenic events. Cytoskeletal remodeling and directional endothelial migration are essential for developmental and pathological angiogenesis. Smad-dependent TGFβ signaling controls vascular patterning and is negatively regulated by microtubules. Here we show that a positive regulator of TGFβ signaling is essential for developmental vascular patterning and microtubule stability. Rudhira/BCAS3 is known to bind microtubules and to play a nodal role in cytoskeletal remodeling and directional endothelial cell (EC) migration in vitro. We demonstrate that the molecular and cellular function of Rudhira is deployed at critical steps in vascular patterning. We generated the first floxed mice for rudhira and find that global or endothelial knockout of rudhira results in mid-gestation lethality due to aberrant embryonic and extra-embryonic vessel patterning and defective cardiac morphogenesis. Rudhira null yolk sac ECs show random and retarded migration. Yolk sac transcriptome analysis revealed key mediators of angiogenic processes and TGFβ receptor signaling were perturbed in rudhira null mutants. Molecular and biochemical analyses showed that rudhira depletion reduced microtubule stability but increased expression of pathway inhibitors leading to high levels of SMAD2/3 ubiquitination and reduced activation. These effects were not rescued by exogenous TGFβ. However, TGFβ treatment of wild type ECs increased Rudhira expression. Further, exogenous Rudhira, which promotes directional cell migration, caused increased SMAD2/3 nuclear translocation and reduced inhibitor levels. Therefore, we propose that Rudhira and TGFβ signaling are mutually dependent. Rudhira has a dual function in promoting TGFβ signaling, possibly by sequestering microtubules and simultaneously preventing SMAD2/3 ubiquitination to permit EC migration and vascular patterning. TGFβ signaling and aberrant human Rudhira (Breast Cancer Amplified Sequence 3, BCAS3) expression are both associated with tumour metastasis. Our study identifies a cytoskeletal, cell type-specific modulator of TGFβ signaling important in development and cancer.

scholarly journals TGFβ receptor internalization into EEA1-enriched early endosomes

2002 ◽  
Vol 158 (7) ◽  
pp. 1239-1249 ◽  
Author(s):  
Susan Hayes ◽  
Anil Chawla ◽  
Silvia Corvera
Keyword(s):  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Type I ◽  
Tgfβ Signaling ◽  
Serine Kinase ◽  
Tgfβ Receptors ◽  
Early Endosomes ◽  
Tgfβ Receptor

Transforming growth factor (TGF)β is an important physiological regulator of cellular growth and differentiation. It activates a receptor threonine/serine kinase that phosphorylates the transcription factor Smad2, which then translocates into the nucleus to trigger specific transcriptional events. Here we show that activated type I and II TGFβ receptors internalize into endosomes containing the early endosomal protein EEA1. The extent of TGFβ-stimulated Smad2 phosphorylation, Smad2 nuclear translocation, and TGFβ-stimulated transcription correlated closely with the extent of internalization of the receptor. TGFβ signaling also requires SARA (Smad anchor for receptor activation), a 135-kD polypeptide that contains a FYVE Zn++ finger motif. Here we show that SARA localizes to endosomes containing EEA1, and that disruption of this localization inhibits TGFβ-induced Smad2 nuclear translocation. These results indicate that traffic of the TGFβ receptor into the endosome enables TGFβ signaling, revealing a novel function for the endosome as a compartment specialized for the amplification of certain extracellular signals.

scholarly journals Skeletal Deformities in Osterix-Cre;Tgfbr2f/f Mice May Cause Postnatal Death

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 975
Author(s):  
Kara Corps ◽  
Monica Stanwick ◽  
Juliann Rectenwald ◽  
Andrew Kruggel ◽  
Sarah B. Peters
Keyword(s):  
Transforming Growth Factor ◽  
Skeletal Development ◽  
Transforming Growth Factor Β ◽  
Tgfβ Signaling ◽  
Skeletal Deformities ◽  
Tooth Abnormalities ◽  
Pathological Analysis ◽  
Diaphragmatic Muscle ◽  
Proliferation And Differentiation ◽  
Tgfβ Receptor

Transforming growth factor β (TGFβ) signaling plays an important role in skeletal development. We previously demonstrated that the loss of TGFβ receptor II (Tgfbr2) in Osterix-Cre-expressing mesenchyme results in defects in bones and teeth due to reduced proliferation and differentiation in pre-osteoblasts and pre-odontoblasts. These Osterix-Cre;Tgfbr2f/f mice typically die within approximately four weeks for unknown reasons. To investigate the cause of death, we performed extensive pathological analysis on Osterix-Cre- (Cre-), Osterix-Cre+;Tgfbr2f/wt (HET), and Osterix-Cre+;Tgfbr2f/f (CKO) mice. We also crossed Osterix-Cre mice with the ROSA26mTmG reporter line to identify potential off-target Cre expression. The findings recapitulated published skeletal and tooth abnormalities and revealed previously unreported osteochondral dysplasia throughout both the appendicular and axial skeletons in the CKO mice, including the calvaria. Alterations to the nasal area and teeth suggest a potentially reduced capacity to sense and process food, while off-target Cre expression in the gastrointestinal tract may indicate an inability to absorb nutrients. Additionally, altered nasal passages and unexplained changes in diaphragmatic muscle support the possibility of hypoxia. We conclude that these mice likely died due to a combination of breathing difficulties, malnutrition, and starvation resulting primarily from skeletal deformities that decreased their ability to sense, gather, and process food.

Characterization of protein production by caprine placental membranes: identification and immunolocalization of retinol-binding protein

1995 ◽  
Vol 146 (3) ◽  
pp. 527-534 ◽  
Author(s):  
K H Liu ◽  
J C Huang ◽  
J D Godkin
Keyword(s):  
Protein Production ◽  
Culture Medium ◽  
De Novo ◽  
Binding Protein ◽  
Yolk Sac ◽  
Retinol Binding Protein ◽  
Minimum Essential Medium ◽  
Placental Membranes ◽  
Extraembryonic Membranes

Abstract Caprine chorion, allantois and amnion from days 23, 28, 35, 39 and 45, and yolk sac from day 23 of pregnancy were isolated by dissection and cultured for 24 h in modified minimum essential medium in the presence of [35S] methionine. De novo-synthesized proteins released into the culture medium were analyzed by two-dimensional PAGE and fluorography. Patterns of protein production by these isolated extraembryonic membranes remained relatively unchanged from days 23 to 45 of pregnancy. Electrophoretic profiles of proteins synthesized by allantois and amnion were identical but distinct from that produced by chorion. Yolk sac was the major source of serum-like proteins. An acidic (pI 5·3–6·3) 22 kDa protein, which consisted of four isoelectric variants, was produced by all extraembryonic membranes and demonstrated to immunoreact with antiserum produced against bovine placental retinol-binding protein (RBP). Limited N-terminal sequence analysis of one major isoform indicated that the protein had complete homology with bovine RBP over the first 15 amino acids. Immunoreactive RBP was localized in epithelial cells lining the chorion, allantois and amnion. In this study, we have characterized and compared protein production by isolated extraembryonic membranes through days 23 to 45 of pregnancy and identified the 22 kDa protein as caprine RBP of placental origin. Journal of Endocrinology (1995) 146, 527–534

scholarly journals Monocyte Subsets with High Osteoclastogenic Potential and Their Epigenetic Regulation Orchestrated by IRF8

2020 ◽  
Author(s):  
Amitabh Das ◽  
Xiaobei Wang ◽  
Jessica Kang ◽  
Alyssa Coulter ◽  
Amol C. Shetty ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Nuclear Translocation ◽  
De Novo ◽  
Primary Source ◽  
Regulatory Elements ◽  
Monocyte Subsets ◽  
Enhancer Activity ◽  
Signaling Receptors ◽  
Early Developmental ◽  
Deficient Mice

SUMMARYOsteoclasts (OCs) are bone resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C− monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of pre-established transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of IRF8, a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8 deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation.

Importance of post-transcriptional regulation of chemokine genes by oxidative stress

2001 ◽  
Vol 360 (2) ◽  
pp. 321-333 ◽  
Author(s):  
Claire JOSSE ◽  
Johan R. BOELAERT ◽  
Martin BEST-BELPOMME ◽  
Jacques PIETTE
Keyword(s):  
Oxidative Stress ◽  
Nuclear Translocation ◽  
De Novo ◽  
Nuclear Factor Κb ◽  
Mitogen Activated Protein Kinase ◽  
U937 Cells ◽  
Transient Transfection Assay ◽  
Mrna Species ◽  
Inflammatory Protein ◽  
Mitogen Activated Protein

The transcription factor, nuclear factor κB (NF-κB), is activated by various stimuli including cytokines, radiation, viruses and oxidative stress. Here we show that, although induction with H2O2 gives rise to NF-κB nuclear translocation in both lymphocyte (CEM) and monocyte (U937) cells, it leads only to the production of mRNA species encoding interleukin-8 (IL-8) and macrophage inflammatory protein 1α in U937 cells. Under similar conditions these mRNA species are not observed in CEM cells. With the use of a transient transfection assay of U937 cells transfected with reporter constructs of the IL-8 promoter and subsequently treated with H2O2, we show that (1) IL-8-promoter-driven transcription is stimulated in both U937 and CEM cells and (2) the NF-κB site is crucial for activation because its deletion abolishes activation by H2O2. The production of IL-8 mRNA in U937 cells is inhibited by the NF-κB inhibitors clasto-lactacystin-β-lactone and E-64D (l-3-trans-ethoxycarbonyloxirane-2-carbonyl-l-leucine-3-methyl amide) but requires protein synthesis de novo. Moreover, inhibition of the p38 mitogen-activated protein kinase also decreases the IL-8 mRNA up-regulation mediated by H2O2. Taken together, these results show the importance of post-transcriptional events controlled by a p38-dependent pathway in the production of IL-8 mRNA in U937. The much lower activation of p38 in CEM cells in response to H2O2 could explain the lack of stabilization of IL-8 mRNA in these cells.

Placenta Is a Niche for Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2671-2671
Author(s):  
Hanna K.A. Mikkola ◽  
Christos Gekas ◽  
Francoise Dieterlen-Lievre ◽  
Stuart H. Orkin
Keyword(s):  
De Novo ◽  
Fetal Liver ◽  
Yolk Sac ◽  
Mouse Development ◽  
Adult Type ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Hematopoietic Organ ◽  
Hematopoietic System ◽  
Hematopoietic Organs

Abstract The hematopoietic system in the embryo develops in anatomically distinct sites, facilitating rapid generation of erythroid cells and formation of a pool of pluripotent HSCs. The origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct the genesis, maturation, expansion and differentiation of HSCs. In avians, de novo hematopoiesis occurs not only in the yolk sac and the AGM but also in another mesodermal appendage, the allantois. In mammals, the allantois forms the umbilical cord and fetal placenta upon fusion with the chorion. The placenta has not been recognized as a hematopoietic organ, although Melchers reported fetal B-cell potential in murine placenta 25 years ago (Nature 1979, 277:219). Recently, Alvarez-Silva et al. showed that the placenta is a rich source for multipotential hematopoietic progenitors prior to the fetal liver (Development2003, 130:5437). We have performed spatial and temporal analysis of HSCs during mouse development and for the first time assessed HSC activity in the placenta. Hematopoietic organs from E10.5-18.5 embryos (CD45.1/CD45.2) were treated with collagenase and transplanted in limiting dilutions (3–1/1000 embryo equivalents, ee) into irradiated CD45.2+ adult hosts with CD45.1+ support BM cells. Reconstitution was analyzed by FACS and HSCs were quantified as repopulating units (RUs/ee = ([reconstituted recipients] /[total recipients]) /[transplanted dose]). Our data show that the placenta functions as a hematopoietic organ that during midgestation harbors a large pool of pluripotent HSCs. The onset of HSC activity in the placenta parallels that of the AGM starting at E10.5–11.0. However, the placenta HSC pool expands until E12.5–13.5 (>50 RUs) contrasting lack of HSC expansion in the AGM. The expansion of CD34+c-kit+ HSCs in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver and is not accompanied with myeloerythroid differentiation. A far greater expansion of placenta HSCs compared to that of clonogenic progenitors (17-fold vs. 2-fold at E11.5–12.5) suggests that the placenta provides a favorable niche for HSCs. Indeed, placenta HSCs possess functional properties of authentic adult-type HSCs by providing high level multilineage reconstitution for >5 months and exhibiting self-renewal capacity upon serial transplantation. Importantly, placenta HSCs are distinct from circulating HSCs that appear in low numbers after E11.5. HSC activity in the placenta declines towards the end of gestation while HSCs in the fetal liver and blood continue to increase, possibly reflecting mobilization of placenta HSCs to the fetal liver and other developing hematopoietic organs. The early onset of HSC activity in the placenta suggests that the allantois and its derivatives may participate in de novo genesis and maturation of HSCs together with the AGM and possibly the yolk sac. As the main blood volume from the dorsal aorta reaches the fetal liver via umbilical vessels and the placenta, placenta may also provide a niche where nascent HSCs, or pre-HSCs, from the AGM colonize for maturation and expansion prior to seeding fetal liver. While further studies are needed to define the precise origin of placenta HSCs and the function of placenta microenvironment as an HSC supportive niche, the unique kinetics and magnitude of HSC activity suggest an important, previously unappreciated role for the placenta in establishing the definitive hematopoietic system.

The Rho GTPase Rac1 Is Not Required for Definitive Hematopoietic Specification in ES-Derived Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1494-1494
Author(s):  
Michael D. Milsom ◽  
Akiko Yabuuchi ◽  
George Q. Daley ◽  
David A. Williams
Keyword(s):  
Rho Gtpase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Embryoid Body ◽  
Es Cells ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitors ◽  
The Impact

Abstract Abstract 1494 Poster Board I-517 Rac1 is a Rho GTPase involved in integrating signaling pathways that regulate numerous cellular processes including adhesion, migration, proliferation and HSC engraftment. Homozygous deletion of Rac1 is lethal in the murine embryo prior to E9.5 and Rac1−/− embryos demonstrate defective gastrulation associated with reduced epiblast adhesion and motility. We have recently demonstrated using lineage-specific conditional deletion that Rac1 insufficiency results in severely impaired hematopoiesis in the embryonic sites of hematopoiesis (AGM, aortic clusters and fetal liver) in the setting of normal hematopoietic development in the yolk sac (YS) and reduced HSC and progenitors in the fetal circulation. This data appears to support the controversial hypothesis that YS derived HSC seed embryonic sites, but an alternative explanation is that Rac1 is essential for some aspect of the induction of intraembryonic hematopoiesis in situ. Another possibility is that Vav1-Cre-mediated excision of Rac1 occurs prior to the onset of hematopoiesis in the embryo proper but not early enough to affect yolk sac hematopoiesis. To test whether Rac1 insufficiency perturbs the normal early differentiation of hematopoietic cells in vitro, we used a lentivirus expressing a Rac1-specific shRNA to knock down expression in an ES line previously characterized to have good hemogenic potential. We observed that the de novo knockdown of Rac1 expression appeared to have no impact upon derivation of hematopoietic progenitors. To demonstrate that this was not the result of inefficient knockdown of Rac1, we derived Rac1−/− ES lines from blastomeres resulting from the mating of Rac1+/− mice. Rac1−/− ES lines were produced in normal Mendelian ratios (4 Rac1+/+: 9 Rac1+/−: 3 Rac1−/−) and did not demonstrate any evidence of abnormal expansion on murine embryonic fibroblasts. In order to assess the impact of Rac1 deficiency on the hemogenic potential of ES cells, standard in vitro differentiation via embryoid body formation was utilized. Neither Rac1 haploinsufficiency nor complete absence of Rac1 had any impact on the production of CD41+/c-Kit+ hematopoietic progenitors within embryoid bodies (Table 1). Furthermore, colony forming assays demonstrated that Rac1 insufficiency did not alter the relative frequency of hematopoietic progenitor compartments (Table 2). We conclude that in the absence of a requirement for vascular migration of HSC, Rac1 is not required for the specification of definitive hematopoiesis. These data, together with our previously published in vivo data continue to support the hypothesis that HSC migration from the YS to the embryo may be required for development of hematopoiesis in the embryo proper. Disclosures: No relevant conflicts of interest to declare.

Occurrence of metachronous pure germinomas long after treatment of a mixed germ cell tumor containing yolk sac tumor and germinoma

2013 ◽  
Vol 11 (1) ◽  
pp. 68-73 ◽  
Author(s):  
Shinya Jinguji ◽  
Kouichirou Okamoto ◽  
Junichi Yoshimura ◽  
Yuichiro Yoneoka ◽  
Ryousuke Ogura ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cell Tumor ◽  
Initial Treatment ◽  
De Novo ◽  
Good Prognosis ◽  
Yolk Sac ◽  
Cell Tumor ◽  
Yolk Sac Tumor ◽  
Mixed Germ Cell Tumor ◽  
Prognosis Group

The authors report a rare case involving the occurrence of metachronous pure germinomas long after treatment of a mixed germ cell tumor (GCT) categorized as having a poor prognosis. A neurohypophysial germinoma occurred 4 years and 6 months after the initial treatment of a mixed pineal GCT containing a yolk sac tumor and a germinoma. Furthermore, intramedullary germinomas occurred 21 years after the initial treatment of the mixed GCT and 15 years after the second treatment of the neurohypophysial germinoma. The neurohypophysial germinoma was not confirmed histopathologically, but the intramedullary germinoma was histopathologically diagnosed as a pure germinoma. Serum α-fetoprotein levels at the second neurohypophysial and third intramedullary occurrences of the germinomas were less than 10 ng/ml. Therefore, no yolk sac components seemed to be contained in the tumors. The second neurohypophysial and third intramedullary germinomas might be recurrences of the germinoma component of the pineal mixed GCT, which consisted of a yolk sac tumor and a germinoma. However, it seems very unlikely that only the germinoma, categorized in the good prognosis group, would be the only one to recur. Hence, it seems plausible that both the second and the third occurrences of pure germinoma were de novo metachronous GCTs arising after the pineal mixed GCT was cured. The authors' case indicates the possibility of multicentric GCTs in the CNS.

scholarly journals Cell-autonomous role of TGFβ and IL-2 receptors in CD4+ and CD8+ inducible regulatory T-cell generation during GVHD

Blood ◽  
2012 ◽  
Vol 119 (23) ◽  
pp. 5575-5583 ◽  
Author(s):  
Norifumi Sawamukai ◽  
Atsushi Satake ◽  
Amanda M. Schmidt ◽  
Ian T. Lamborn ◽  
Priti Ojha ◽  
...  
Keyword(s):  
T Cells ◽  
Potential Role ◽  
Acute Gvhd ◽  
De Novo ◽  
Receptor Expression ◽  
Tgfβ Receptor ◽  
Bona Fide ◽  
Deficient Mice ◽  
Transplantation Recipient

Abstract FoxP3+ regulatory T cells (Tregs) suppress GVHD while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from the expansion of preexisting natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor Tconvs in the transplantation recipient. Using an MHC-mismatched model of acute GVHD, in the present study we found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Experiments using various combinations of T cells from wild-type and FoxP3-deficient mice suggested that both preexisting donor nTregs and the generation of iTregs in the recipient mice contribute to protection against GVHD. Surprisingly, CD8+FoxP3+ T cells represented approximately 70% of the iTreg pool. These CD8+FoxP3+ T cells shared phenotypic markers with their CD4+ counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4+ and CD8+ Tregs appeared to be protective against GVHD-induced lethality and required IL-2 and TGFβ receptor expression for their generation. These data illustrate the complex makeup of the donor-derived FoxP3+ Treg pool in allogeneic recipients and their potential role in protection against GVHD.

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