Erythroid Cells Adapt to L-Leucine Scarcity By Reducing Hemoglobin Production Via the mTORC1/4E-BP Pathway

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2660-2660
Author(s):  
Jacky Chung ◽  
Daniel E. Bauer ◽  
Alireza Ghamari ◽  
Christopher P. Nizzi ◽  
Kathryn M. Deck ◽  
...  
Keyword(s):  
Normal Development ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Regulatory Mechanisms ◽  
Erythroid Cells ◽  
Amino Acid Deprivation ◽  
General Control ◽  
Leucine Uptake ◽  
Multicellular Organisms ◽  
Hemoglobin Production

Abstract In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. Here, we find that maturing erythroid cells increase L-leucine uptake via transcriptional up-regulation of the L-leucine transporter, LAT3. Inadequate L-leucine uptake by L-leucine starvation or LAT3 inhibition triggers a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells via the mTORC1/4E-BP pathway. CRISPR-mediated deletion of 4E-BPs in murine erythroid cells renders them resistant to mTORC1 and LAT3 inhibition, markedly restoring hemoglobinization. Our complementary results demonstrate that globins are direct translational mTORC1 targets during normal development. This pathway is distinct from the previously reported translational regulatory mechanisms mediated by the heme-regulated inhibitor (HRI) kinase or by severe amino acid deprivation via the general control nonderepressible 2 (GCN2) kinase. We propose that, in red cells, mTORC1 serves as a homeostatic sensor coupling hemoglobin production to sufficient L-leucine uptake. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Regulation Of Erythro-Megakaryocytic Lineage Bifurcation By The Gfi1b Gene Target Rgs18

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1191-1191
Author(s):  
Ananya Sengupta ◽  
Ghanshyam Upadhyay ◽  
Asif Chowdhury ◽  
Shireen Saleque
Keyword(s):  
Conflicts Of Interest ◽  
Integration Site ◽  
Fetal Liver ◽  
Cell Types ◽  
Mapk Signaling ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Gene Target ◽  
Over Expression ◽  
In Erythroid Cells

Abstract The molecular basis for the divergence of the erythroid (red blood cell) and megakaryocyte (platelet) lineages from a common bipotent MEP (megakaryocyte-erythroid progenitor) remains undefined. We now demonstrate that Rgs18 (regulator of G protein signaling 18), a GAP (GTPase activating protein) factor and a transcriptional gene target of the Gfi1b transcriptional repressor complex, likely arbitrates this critical lineage decision downstream of Gfi1b. Rgs18 was identified in a chromatin immunoprecipitation (ChIP on chip) screen for Gfi1b/LSD1/Rcor1 targets in erythroid cells. Accordingly, Rgs18 expression was found to be up-regulated in LSD1 inhibited, and Gfi1b deficient erythroid cells confirming repression of this gene by Gfi1b and its co-factors in this lineage. In contrast, Rgs18 expression was comparable in megakaryocytic cells derived from wild type and gfi1b-/-hematopoietic progenitors indicating Gfi1b independent expression of Rgs18 in these cells. Manipulation of Rgs18 expression produced opposite effects in the erythroid and megakaryocytic lineages. Rgs18 inhibition retarded megakaryocytic differentiation while its ectopic over-expression promoted differentiation at the expense of proliferation. The reverse was observed in erythroid cells where Rgs18 inhibition produced an enhancement of differentiation while over-expression impaired erythropoiesis. Since Rgs signaling regulates the activity of downstream MAPK pathways we determined the status of these pathways in Rgs18 manipulated cells. Inhibition of Rgs18 stimulated ERK phosphorylation in megakaryocytes but diminished it in erythroid cells. In contrast, Rgs18 inhibition in erythroid cells elevated p38MAPK protein and phosphorylation levels. The opposite effects of Rgs18 manipulation on MAPK signaling in erythroid versus megakaryocytic cells while intriguing are consistent with the changes in differentiation and proliferation observed in each lineage, respectively. Although Rgs18 manipulation produced opposite effects in erythroid and megakaryocytic cells, the level and activity of this factor correlated similarly with those of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1) and KLF1/EKLF (Kruppel like factor1) in both cell types. In both lineages, Rgs18 protein levels correlated directly with Fli1, and inversely with KLF1, message levels. Since Fli1 promotes megakaryocytic, and KLF1 erythroid, development; these results demonstrate that Rgs18 promotes the emergence of megakaryocytic cells from bipotent MEPs by modulating MAPK signaling and altering Fli1/KLF1 stoichiometries. Although it is unclear why Gfi1b mediated repression of Rgs18 is erythroid specific even though the former is expressed in both lineages, these results demonstrate that repression of Rgs18 by Gfi1b in fetal liver MEPs limits megakaryopoiesis and augments erythropoiesis. However following megakaryocytic commitment, robust Gfi1b independent expression of Rgs18 drives differentiation of this lineage while continued repression of Rgs18 by Gfi1b in erythroid cells ensures their proper maturation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Erythropoietin Signaling Regulates Heme Biosynthesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 543-543
Author(s):  
Jacky Chung ◽  
Johannes G. Wittig ◽  
Alireza Ghamari ◽  
Manami Maeda ◽  
Harvey Lodish ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Protoporphyrin Ix ◽  
Janus Kinase ◽  
Heme Biosynthesis ◽  
Erythroid Cells ◽  
Downstream Target ◽  
Hematologic Disorder ◽  
Hemoglobin Production ◽  
Dependent Mechanism

Abstract Heme plays a fundamental role in a diverse array of cellular processes and is required for the survival of all cells. During erythropoiesis, heme production is drastically upregulated to support the production of oxygen-carrying hemoglobin. This increase in heme production is mediated by transcriptional induction of heme metabolism genes including ferrochelatase (FECH), which is the enzyme that catalyzes the rate-limiting insertion of ferrous iron into protoporphyrin IX in the mitochondria of erythroid cells. However, how heme production is coordinately regulated by extracellular cues is currently unknown. Here, using complementary biochemical and genetics approaches, we show that erythropoietin (EPO) signaling regulates heme biosynthesis via a protein kinase A (PKA)-dependent mechanism. In its inactive state, PKA is a tetrameric complex consisting of two catalytic subunits (C) that are bound to and inhibited by two regulatory subunits (R). The C subunits become activated to phosphorylate downstream target proteins when they dissociate from the R subunits. We demonstrate that EPO-induced JAK2 (janus kinase 2) activity leads to release of the C subunits from the R subunits. We also find that phosphorylated STAT5 (signal transducer and activator of transcription 5) forms a molecular complex with PKA-C. This suggests that phospho-STAT5 can outcompete PKA-R to release PKA-C to directly phosphorylate FECH at a highly conserved threonine residue located in the catalytic site. We examined the importance of FECH phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. Erythroid cells harboring the homozygous Thr115Ala Fech mutation exhibited a block in hemoglobin production and concomitant intracellular accumulation of upstream protoporphyrin intermediates. Strikingly, this phenotype bears resemblance to erythropoietic protoporphyria (EPP), a human hematologic disorder typically associated with FECHmutations. Together, our results support a model where EPO signaling during erythroid maturation activates PKA by a previously unrecognized JAK2/STAT5-dependent mechanism. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Furthermore, our data implicates aberrant EPO/JAK2/PKA signaling in the pathogenesis of human EPP. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Multiple Apoptotic Defects In Hematopoietic Cellsfrom Mice Lacking Lipocalin 24p3.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1560-1560
Author(s):  
L.R. Devireddy ◽  
Zhuoming Liu ◽  
Alieta Ciocea ◽  
Amy Yang ◽  
Zhengqi Wang ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Cell Types ◽  
Erythroid Cells ◽  
General Role ◽  
Physiological Processes ◽  
Iron Trafficking ◽  
Apoptotic Activity ◽  
Induced Apoptosis

Abstract Abstract 1560 The lipocalin mouse 24p3 has been implicated in diverse physiological processes including apoptosis, iron trafficking, development and innate immunity. Studies from our lab as well as others demonstrated the pro-apoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the homeostasis of the hematopoietic system has not been tested in vivo. To study the role of 24p3 in apoptosis, we derived 24p3 null mice on 129 SVE and C57BL/6 genetic backgrounds, which enabled us to test, for the first time, the in vivo role of 24p3 without any confounders. We find that homozygous 24p3-/- mice have apoptotic defects in several hematopoietic lineages, including neutrophils, cytokine-dependent mast cells, thymocytes and erythroid cells. The progressive accumulation of lymphoid, myeloid and erythroid cells in 24p3-/- mice demonstrate the importance of 24p3 in the regulation of homeostasis of hematopoietic cells in adults. Thymocytes isolated from 24p3 null mice displayed resistance to apoptosis-induced by Dexamethasone. In addition, Dexamethasone-induced apoptosis is also reduced in 24p3-/- mice. Collectively, these results indicate that 24p3 is an important target gene involved in GC-mediated apoptosis of lymphocytes and other hematopoietic cell types. Finally, our results also indicate that the genetic background of mice can modulate the effects of 24p3 deficiency. In summary, the results presented here, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Disclosures: No relevant conflicts of interest to declare.

Role of Transcriptional Read-Through in PRE Activity in Drosophila melanogaster

Acta Naturae ◽  
2016 ◽  
Vol 8 (2) ◽  
pp. 79-86 ◽  
Author(s):  
P. V. Elizar’ev ◽  
D. V. Lomaev ◽  
D. A. Chetverina ◽  
P. G. Georgiev ◽  
M. M. Erokhin
Keyword(s):  
Dna Sequences ◽  
Cell Types ◽  
Transcription Terminator ◽  
Response Elements ◽  
Polycomb Response Elements ◽  
The Individual ◽  
Multicellular Organisms ◽  
Different Cell Types ◽  
Main Factor

Maintenance of the individual patterns of gene expression in different cell types is required for the differentiation and development of multicellular organisms. Expression of many genes is controlled by Polycomb (PcG) and Trithorax (TrxG) group proteins that act through association with chromatin. PcG/TrxG are assembled on the DNA sequences termed PREs (Polycomb Response Elements), the activity of which can be modulated and switched from repression to activation. In this study, we analyzed the influence of transcriptional read-through on PRE activity switch mediated by the yeast activator GAL4. We show that a transcription terminator inserted between the promoter and PRE doesnt prevent switching of PRE activity from repression to activation. We demonstrate that, independently of PRE orientation, high levels of transcription fail to dislodge PcG/TrxG proteins from PRE in the absence of a terminator. Thus, transcription is not the main factor required for PRE activity switch.

scholarly journals Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jillian L. Lindblad ◽  
Meghana Tare ◽  
Alla Amcheslavsky ◽  
Alicia Shields ◽  
Andreas Bergmann
Keyword(s):  
Normal Development ◽  
Cell Lineage ◽  
Cell Types ◽  
Specific Role ◽  
Mutant Phenotype ◽  
Apoptotic Pathway ◽  
Specific Expression ◽  
Card Domain

AbstractThe initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc+ in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.

Artificial metaplasia of pigmented epithelium into retina in tadpoles and adult frogs

Development ◽  
1972 ◽  
Vol 28 (3) ◽  
pp. 521-546
Author(s):  
G. V. Lopashov ◽  
Alla A. Sologub
Keyword(s):  
Rana Temporaria ◽  
Cell Types ◽  
Control Mechanisms ◽  
General Control ◽  
Pigmented Epithelium ◽  
The Stability

The present work was aimed at investigating the possibility and conditions necessary for the artificial transformation of one tissue into another. Experiments were carried out with the pigmented epithelium of the eye in tadpoles and adult frogs of Rana temporaria. Following the removal of the mesenchyme envelopes (or their exfoliation during the experiment), pigmented epithelium transformed into retina under the influence of retina from tadpoles of the same species. This phenomenon was observed both under the cultivation of a piece of retina in a sandwich of pigmented epithelium and the transplantation of pigmented epithelium layers into the eye cavity of tadpoles. Such transformation did not occur in the absence of retinal influence. Metaplasia requires the removal of the mesenchyme envelopes, the action of the retinal agent, as well as preservation of the integrity of the pigmented epithelium layer and subsequent proliferation of its cells. The character of general control mechanisms both maintaining the stability of cell types and leading to their transformation into other cell types is discussed.

scholarly journals Fra-2 negatively regulates postnatal alveolar septation by modulating myofibroblast function

2017 ◽  
Vol 313 (5) ◽  
pp. L878-L888 ◽  
Author(s):  
Kazuyuki Tsujino ◽  
John T. Li ◽  
Tatsuya Tsukui ◽  
Xin Ren ◽  
Latifa Bakiri ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Fibrosis ◽  
Vascular Remodeling ◽  
Normal Development ◽  
Smooth Muscle Actin ◽  
Cell Types ◽  
Lineage Tracing ◽  
Mutant Mice ◽  
Pulmonary Vascular Remodeling ◽  
Migration Capacity

Mice that globally overexpress the transcription factor Fos-related antigen-2 (Fra-2) develop extensive pulmonary fibrosis and pulmonary vascular remodeling. To determine if these phenotypes are a consequence of ectopic Fra-2 expression in vascular smooth muscle cells and myofibroblasts, we generated mice that overexpress Fra-2 specifically in these cell types (α-SMA-rtTA;tetO-Fra-2). Surprisingly, these mice did not develop vascular remodeling or pulmonary fibrosis but did develop a spontaneous emphysema-like phenotype characterized by alveolar enlargement. Secondary septa formation is an important step in the normal development of lung alveoli, and α-smooth muscle actin (SMA)-expressing fibroblasts (myofibroblasts) play a crucial role in this process. The mutant mice showed reduced numbers of secondary septa at postnatal day 7 and enlarged alveolae starting at postnatal day 12, suggesting impairment of secondary septa formation. Lineage tracing using α-SMA-rtTA mice crossed to a floxed TdTomato reporter revealed that embryonic expression of α-SMA Cre marked a population of cells that gave rise to nearly all alveolar myofibroblasts. Comprehensive transcriptome analyses (RNA sequencing) demonstrated that the overwhelming majority of genes whose expression was significantly altered by overexpression of Fra-2 in myofibroblasts encoded secreted proteins, components of the extracellular matrix (ECM), and cell adhesion-associated genes, including coordinate upregulation of pairs of integrins and their principal ECM ligands. In addition, primary myofibroblasts isolated from the mutant mice showed reduced migration capacity. These findings suggest that Fra-2 overexpression might impair myofibroblast functions crucial for secondary septation, such as myofibroblast migration across alveoli, by perturbing interactions between integrins and locally produced components of the ECM.

scholarly journals In vivotissue-specific chromatin profiling inDrosophila melanogasterusing GFP-tagged nuclei

2021 ◽  
Author(s):  
Juan Jauregui-Lozano ◽  
Kimaya Bakhle ◽  
Vikki M. Weake
Keyword(s):  
Cell Types ◽  
Chromatin Accessibility ◽  
Chromatin State ◽  
Specific Cell ◽  
Histone Marks ◽  
The Many ◽  
Chromatin Profiling ◽  
Photoreceptor Neurons ◽  
Multicellular Organisms

AbstractThe chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasisin vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolateDrosophilanuclei tagged with GFP expressed under Gal4/UAS control. Using this protocol, we profiled chromatin accessibility using Omni-ATAC, and examined the distribution of histone marks using ChIP-seq and CUT&Tag in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types inDrosophila.

scholarly journals Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis

2021 ◽  
Vol 12 (12) ◽  
Author(s):  
Hyeon-Ok Jin ◽  
Sung-Eun Hong ◽  
Ji-Young Kim ◽  
Se-Kyeong Jang ◽  
In-Chul Park
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Survival ◽  
Amino Acid Deprivation ◽  
General Control ◽  
Akt Activation ◽  
Amino Acid Availability ◽  
Survival Signal ◽  
Cancer Cell Survival ◽  
Glutamine Deprivation

AbstractAmino acid availability is sensed by various signaling molecules, including general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). However, it is unclear how these sensors are associated with cancer cell survival under low amino acid availability. In the present study, we investigated AKT activation in non-small cell lung cancer (NSCLC) cells deprived of each one of 20 amino acids. Among the 20 amino acids, deprivation of glutamine, arginine, methionine, and lysine induced AKT activation. AKT activation was induced by GCN2/ATF4/REDD1 axis-mediated mTORC2 activation under amino acid deprivation. In CRISPR-Cas9-mediated REDD1-knockout cells, AKT activation was not induced by amino acid deprivation, indicating that REDD1 plays a major role in AKT activation under amino acid deprivation. Knockout of REDD1 sensitized cells cultured under glutamine deprivation conditions to radiotherapy. Taken together, GCN2/ATF4/REDD1 axis induced by amino acid deprivation promotes cell survival signal, which might be a potential target for cancer therapy.

The developmentally regulated ECM glycoprotein ISG plays an essential role in organizing the ECM and orienting the cells of Volvox

2000 ◽  
Vol 113 (24) ◽  
pp. 4605-4617
Author(s):  
A. Hallmann ◽  
D.L. Kirk
Keyword(s):  
Critical Role ◽  
Somatic Cells ◽  
Cell Types ◽  
Model System ◽  
Swimming Behavior ◽  
Truncated Form ◽  
Developmentally Regulated ◽  
Gene Encoding ◽  
Ecm Architecture ◽  
Multicellular Organisms

Volvox is one of the simplest multicellular organisms with only two cell types, yet it has a surprisingly complex extracellular matrix (ECM) containing many region-specific morphological components, making Volvox suitable as a model system for ECM investigations. ECM deposition begins shortly after inversion, which is the process by which the embryo turns itself right-side-out at the end of embryogenesis. It was previously shown that the gene encoding an ECM glycoprotein called ISG is transcribed very transiently during inversion. Here we show that the developmentally controlled ISG accumulates at the bases of the flagella right after inversion, before any morphologically recognizable ECM structures have yet developed. Later, ISG is abundant in the ‘flagellar hillocks’ that encircle the basal ends of all flagella, and in the adjacent ‘boundary zone’ that delimits the spheroid. Transgenic Volvox were generated which express a truncated form of ISG. These transgenics exhibit a severely disorganized ECM within which the cells are embedded in a highly chaotic manner that precludes motility. A synthetic version of the C-terminal decapeptide of ISG has a similar disorganizing effect, but only when it is applied during or shortly after inversion. We postulate that ISG plays a critical role in morphogenesis and acts as a key organizer of ECM architecture; at the very beginning of ECM formation ISG establishes an essential initial framework that both holds the somatic cells in an adaptive orientation and acts as the scaffold upon which the rest of the ECM can be properly assembled, assuring that somatic cells of post-inversion spheroids are held in orientations and locations that makes adaptive swimming behavior possible.

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