A Screen In Primary Erythroblasts Reveals a MicroRNA-Centered Homeostatic Mechanism for Regulating Cyclin E Activity.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1543-1543
Author(s):  
Yanfei Xu ◽  
Tanushri Sengupta ◽  
Alexander C. Minella
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
Conflicts Of Interest ◽  
Cyclin E ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Homeostatic Mechanism ◽  
Regulated Expression

Abstract Abstract 1543 A growing body of evidence highlights the importance of microRNAs in regulating the expression of mediators of cell cycle progression. A theme emerging from these studies is that microRNAs participate in feedback or feed-forward circuits to provide bistability for key transition points in the cell cycle. We previously have shown that proper regulation of cyclin E activity is required for normal erythroid cell maturation in vivo, using cyclin ET74AüT393A knock-in mice, which have markedly dysregulated cyclin E due to its failure to interact with the Fbw7 ubiquitin ligase complex. We hypothesized that we could identify novel, microRNA-based molecular circuitry for maintaining appropriate levels of cyclin E activity by screening cyclin E knock-in erythroblasts for alterations in microRNA expression. We analyzed data we obtained from multiplex real-time PCR arrays comparing the expression of over 500 microRNAs in cyclin ET74A T393A knock-in versus wild-type erythroblasts (Ter119+/CD71+) and found down-regulated expression of a number of microRNAs targeting CDK inhibitors. We also identified down-regulated expression of potential microRNA regulators of Fbw7 expression. We found that overexpression of miR-223, in particular, significantly reduces Fbw7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the Fbw7 3’ untranslated region. We then found that reduced miR-223 expression leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression in K562 cells is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway and that dysregulated Fbw7 expression alters the erythroid differentiation capacity of these cells. Mir-223 plays an important role in myeloid and erythroid differentiation by regulating multiple substrates involved in these maturation programs. Here, we identify Fbw7 as a novel target of miR-223. Our data also indicate that miR-223 modulates Fbw7 expression as part of a homeostatic mechanism to regulate cyclin E activity and provide the first evidence that activity of the SCFFbw7 ubiquitin ligase can be controlled by the microRNA pathway. Disclosures: No relevant conflicts of interest to declare.

Cyclin E Regulation by the Fbw7 Ubiquitin Ligase Pathway Is Required for Normal Mitophagy and Restraining Reactive Oxidative Species During Erythroid Cell Maturation In Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3393-3393
Author(s):  
Yanfei Xu ◽  
Ka Tat Siu ◽  
Amit Verma ◽  
Alexander C. Minella
Keyword(s):  
Ubiquitin Ligase ◽  
Conflicts Of Interest ◽  
Cyclin E ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Reactive Oxidative Species ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Oxidative Species

Abstract Abstract 3393 We previously described a knock-in mouse model that permits study of the physiologic consequences of cyclin E deregulation, by selective ablation of its regulation via the SCFFbw7 ubiquitin ligase. We found that erythroid progenitor cells in our cyclin ET74A T393A knock-in mice exhibit abnormally increased proliferation, increased apoptosis, impaired maturation, and dysplastic morphologies. Most prominent among the gene expression alterations we have identified in the cyclin E knock-in erythroid cells is induction of multiple p53 target genes, consistent with p53 pathway activation. In contrast to several recently described models of ribosomal protein gene mutations, in which p53 activation appears to induce dyserythropoiesis, we determined that p53 function actually maintains partially compensated erythroid cell maturation in vivo, in the context of impaired Fbw7-mediated cyclin E degradation. We next found that dysregulated cyclin E-CDK2 activity in cyclin ET74A T393A erythroid cells is associated with increased reactive oxidative species and increased mitochondrial mass and activity. These results coincide with findings of abnormal mitochondria retention in late-stage erythroid cells and significantly down-regulated expression of BNIP3L (NIX). BNIP3L encodes a critical regulator of erythroid cell mitophagy, and the transcriptional controls maintaining its expression in maturing erythroid cells likely account for why this lineage is acutely sensitive to deregulated cyclin E activity. Finally, we show evidence that reduced expression of BNIP3L may play a role in some cases of early-stage myelodysplastic syndromes. Disclosures: No relevant conflicts of interest to declare.

Regulation of Erythropoiesis by Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin A (DZNep)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 982-982
Author(s):  
Tohru Fujiwara ◽  
Haruka Saitoh ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Wide Range ◽  
The Impact ◽  
Chip Analysis

Abstract Abstract 982 Background. EZH2, a core component of Polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through mediating trimethylation of histone H3 at lysine 27 (H3K27), and is involved in various biological processes, including hematopoiesis. Overexpression of EZH2 has been identified in a wide range of solid tumors as well as hematological malignancies. Recent studies indicated that 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, preferentially induces apoptosis in cancer cells, including acute myeloid leukemia and myelodysplastic syndromes, implying that EZH2 may be a potential new target for epigenetic treatment. On the other hand, whereas PRC2 complex has been reported to participate in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation (Yu et al. Mol Cell 2009; Ross et al. MCB 2012), the impact of DZNep on erythropoiesis has not been evaluated. Method. The K562 erythroid cell line was used for the analysis. The cells were treated with DZNep at doses of 0.2 and 1 microM for 72 h. Quantitative ChIP analysis was performed using antibodies to acetylated H3K9 and GATA-1 (Abcam). siRNA-mediated knockdown of EZH2 was conducted using Amaxa nucleofection technology™ (Amaxa Inc.). For transcription profiling, SurePrint G3 Human GE 8 × 60K (Agilent) and Human Oligo chip 25K (Toray) were used for DZNep-treated and EZH2 knockdown K562 cells, respectively. Gene Ontology was analyzed using the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/). Results. We first confirmed that DZNep treatment decreased EZH2 protein expression without significantly affecting EZH2 mRNA levels, suggesting that EZH2 was inhibited at the posttranscriptional level. We also confirmed that DZNep treatment significantly inhibited cell growth. Interestingly, the treatment significantly induced erythroid differentiation of K562 cells, as determined by benzidine staining. Transcriptional profiling with untreated and DZNep-treated K562 cells (1 microM) revealed that 789 and 698 genes were upregulated and downregulated (> 2-fold), respectively. The DZNep-induced gene ensemble included prototypical GATA-1 targets, such as SLC4A1, EPB42, ALAS2, HBA, HBG, and HBB. Concomitantly, DZNep treatment at both 0.2 and 1 microM upregulated GATA-1 protein level as determined by Western blotting, whereas the effect on its mRNA levels was weak (1.02- and 1.43-fold induction with 0.2 and 1 microM DZNep treatment, P = 0.73 and 0.026, respectively). Furthermore, analysis using cycloheximide treatment, which blocks protein synthesis, indicated that DZNep treatment could prolong the half-life of GATA-1 protein, suggesting that DZNep may stabilize GATA-1 protein, possibly by affecting proteolytic pathways. Quantitative ChIP analysis confirmed significantly increased GATA-1 occupancy as well as increased acetylated H3K9 levels at the regulatory regions of these target genes. Next, to examine whether the observed results of DZNep treatment were due to the direct inhibition of EZH2 or hitherto unrecognized effects of the compound, we conducted siRNA-mediated transient knockdown of EZH2 in K562 cells. Quantitative RT-PCR analysis demonstrated that siRNA-mediated EZH2 knockdown had no significant effect on the expression of GATA-1 as well as erythroid-lineage related genes. Furthermore, transcription profiles of the genes in the quantitative range of the array were quite similar between control and EZH2 siRNA-treated K562 cells, with a correlation efficient of 0.977. Based on our profiling results, we are currently exploring the molecular mechanisms by which DZNep promotes erythroid differentiation of K562 cells. Conclusion. DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism not directly related to EZH2 inhibition. Our microarray analysis of DZNep-treated K562 cells may provide a better understanding of the mechanism of action of DZNep. Disclosures: No relevant conflicts of interest to declare.

A Mouse Model of Hematopoietic Stem Cell Failure Associated with p53-Loss and Defective Fbw7-Dependent Cyclin E Regulation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2297-2297
Author(s):  
Ka Tat Siu ◽  
Yanfei Xu ◽  
Mitra Bhattacharyya ◽  
Alexander C. Minella
Keyword(s):  
Cell Cycle ◽  
Mouse Model ◽  
Conflicts Of Interest ◽  
Cyclin E ◽  
Cell Cycle Control ◽  
Control Mechanisms ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 2297 Recent findings have challenged the notion that increased proliferation of hematopoietic stem cells (HSCs) necessarily restricts their self-renewal capacity. We have studied the physiologic consequences to HSCs of ablating a key cell cycle regulatory mechanism, Fbw7-dependent cyclin E ubiquitination, using germline knock-in of a cyclin ET74A T393A allele. Fbw7 is a tumor suppressor that regulates the abundance of several oncoprotein substrates by ubiquitin-mediated proteolysis, including cyclin E, Notch, and c-Myc. Cyclin E overexpression in vivo is associated with increased proliferation in some cellular contexts as well as a variety of deleterious consequences, including genomic instability, senescence, or apoptosis. In HSCs, Fbw7-loss has been shown to induce self-renewal and multi-lineage reconstitution defects, and the effect of Fbw7-loss in HSCs has been ascribed to dysregulated Myc and Notch expression. Using the cyclin ET74A T393A mouse model, we tested the hypothesis that impaired Fbw7-mediated regulation of cyclin E, specifically, promotes HSC exhaustion due to loss of self-renewal capacity. We first examined bone marrow HSC counts and their cell cycle kinetics in cyclin E knock-in and wild-type control mice at steady state and following hematologic injury induced by 5-fluorouracil treatment. We found that cyclin E dysregulation reduces numbers of quiescent HSCs and increases cells in S/G2/M-phases, while decreasing total numbers of HSCs, phenotypes made more severe after recovery from hematologic stress. Using bromodeoxyuridine labeling studies, we found that excess cyclin E activity causes DNA hyper-replication in cyclin ET74A T393A HSCs in a cell autonomous manner. By enumerating multi-potent progenitors (MPPs), we ruled out increased rate of transit from HSC-to-MPP as a cause of the apparent exhaustion of cyclin E knock-in HSCs. Thus, dysregulated cyclin E in HSCs promotes both increased proliferation and depletion of the HSC pool. Serial transplantation further revealed peripheral blood reconstitution defects associated with cyclin ET74A T393A HSCs. Recently, we have found that p53 is activated by dysregulated cyclin E in hematopoietic cells in vivo, in association with phosphorylation of both p53 and Chk1 proteins, resembling a DNA damage-type response. Interestingly, p53-loss has been found to be associated with a gain of HSC self-renewal activity. We therefore hypothesized that p53-loss would rescue the self-renewal defect of cyclin E knock-in HSCs. Surprisingly, we discovered that cyclin ET74A T393A; p53-null HSCs showed evidence of significantly worse self-renewal and peripheral reconstitution, compared to p53-null HSCs, defects that are more severe than those associated with impaired Fbw7-mediated cyclin E control in the setting of wild-type p53 (Chi-squared test, p<0.0001). Thus, our data are consistent with the concept that intact p53 function, in the setting of oncogenic insult, can preserve partial HSC self-renewal capacity, and its loss in vivo is detrimental to HSC viability when accompanied by defects in cell cycle control mechanisms. Disclosures: No relevant conflicts of interest to declare.

Chromosome Instability Underlies Stem Cell Dysfunction and Neoplasia In Widespread Hematologic Disease Induced By Deregulated Cyclin E

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 220-220
Author(s):  
Ka Tat Siu ◽  
Yanfei Xu ◽  
Mitra Bhattacharyya ◽  
Sandeep Gurbuxani ◽  
Youjia Hua ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Conflicts Of Interest ◽  
Cyclin E ◽  
The Self ◽  
Comparative Genome Hybridization ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cyclin E1 ◽  
Serial Transplantation

Abstract The Fbw7 ubiquitin ligase controls the expression of a number of oncoprotein substrates including cyclin E, Notch, c-Jun, and c-Myc. Using a knock-in mouse model (cyclin ET74AT393A), in which mutations were introduced into the cyclin E1 allele (Ccne1) to disrupt Fbw7-mediated ubiquitination specifically, we previously found that cyclin E dysregulation in vivo induces anemia with defects in erythroid differentiation and morphologic dysplasia of erythroid progenitors. We also found that cyclin ET74AT393A mice have fewer hematopoietic stem cells (HSCs) during steady-state hematopoiesis compared to wild-type counterparts. We performed serial transplantation experiments to assay comprehensively the self-renewal and multi-lineage reconstitution capacities of cyclin ET74AT393A HSCs. Contrary to our expectations, cyclin ET74AT393A HSC self-renewal appears normal after three rounds of serial transplantation; however, we identified defects in their multi-lineage reconstitution function. In cyclin ET74A T393A bone marrow erythroid cells, induction of a p53-dependent DNA damage response pathway appears to promote compensated erythropoiesis. In cyclin ET74A T393A HSCs, we similarly observed induced expression of canonical p53 target genes. We studied the effect of p53-loss on cyclin ET74A T393A HSCs and found that p53-null; cyclin ET74A T393A HSCs exhibit defects in both self-renewal and multi-lineage reconstitution. By enumerating chromosomes in metaphase spreads, we found p53-null; cyclin ET74A T393A hematopoietic stem and progenitor cells (HSPCs) demonstrate significant chromosomal instability (CIN). Importantly, we can recapitulate the self-renewal defects and CIN of cyclin ET74A T393A HSPCs with intact p53 by treating recipient animals with a single dose of 5-fluorouracil (5-FU). Thus, chromosomal stability is a key determinant for the maintenance of HSC self-renewal, and hematologic stress appears to unmask the potential for impaired Fbw7-dependent cyclin E ubiquitination to engender CIN in the presence of intact p53. Moreover, CIN is a characteristic feature of fatal T-cell malignancies that ultimately develop in recipients of cyclin ET74A T393A; p53-null HSCs. In pre-malignant thymocytes isolated from recipients of cyclin ET74A T393A; p53-null HSCs, aneuploidy is associated with the marked potentiation of cyclin E kinase activity in these cells by p53-loss. In malignant thymocytes, comparative genome hybridization analysis demonstrates clonal CIN associated with deregulated cyclin E expression combined with p53-loss. In toto, our data demonstrate the functional importance of cyclin E regulation by the Fbw7 ubiquitin ligase to the hematopoietic system and highlight CIN as a key mechanism underlying HSC dysfunction and malignancy induced by deregulated cyclin E in vivo. Disclosures: No relevant conflicts of interest to declare.

Cell cycle regulation of a mouse histone H4 gene requires the H4 promoter

1987 ◽  
Vol 7 (3) ◽  
pp. 1048-1054
Author(s):  
A Seiler-Tuyns ◽  
B M Paterson
Keyword(s):  
Cell Cycle ◽  
Shuttle Vector ◽  
Constitutive Expression ◽  
Mrna Levels ◽  
Histone H4 ◽  
Coding Region ◽  
Regulated Expression ◽  
Arrest Growth ◽  
Globin Promoter ◽  
Histone H4 Gene

The mouse histone H4 gene, when stably transformed into L cells on the PSV2gpt shuttle vector, is cell cycle regulated in parallel with the endogenous H4 genes. This was determined in exponentially growing pools of transformants fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cell cycle without the use of treatments that arrest growth. Linker additions in the 5' noncoding region of the H4 RNA or in the coding region of the gene did not affect the cell cycle-regulated expression of the modified H4 gene even though the overall level of expression was altered. However, replacing the H4 promoter with the human alpha-2 globin promoter, so that the histone transcript produced by the chimeric gene remains essentially unchanged, resulted in the constitutive expression of H4 mRNA during all phases of the cell cycle with no net increase in H4 mRNA levels during the G1-to-S transition. From these results we conclude that all the information necessary for the cell cycle-regulated expression of the H4 gene is contained in the 5.2-kilobase subclone used in these studies with 228 nucleotides of 5'-flanking DNA and that the increase in H4 mRNA during the G1-to-S transition in the cell cycle is mediated by the H4 promoter and not by the increased stability of the H4 RNA.

scholarly journals Mediation of the inhibitory effect of thyroid hormone on proliferation of hepatoma cells by transforming growth factor-beta

2006 ◽  
Vol 36 (1) ◽  
pp. 9-21 ◽  
Author(s):  
Chun-Che Yen ◽  
Ya-Hui Huang ◽  
Chu-Yu Liao ◽  
Cheng-Jung Liao ◽  
Wan-Li Cheng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Thyroid Hormone ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Transforming Growth Factor ◽  
Cyclin E ◽  
Hepatoma Cell ◽  
Stable Cell Line ◽  
Mrna Levels

Thyroid hormone (triiodothyronine, T3) regulates growth, development and differentiation. To examine the influence of T3 on hepatoma cell growth, thyroid receptor (TR)α1 or TRβ1 over-expressing HepG2 cell lines were used. Growth of the HepG2-TR stable cell line was inhibited by over 50% following treatment with T3. However, transforming growth factor (TGF)-β neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3. Flow cytometric analysis indicated that the growth inhibition was apparent at the transition point between the G1 and S phases of the cell cycle. The expression of major cell cycle regulators was used to provide further evidence for the growth inhibition. Cyclin-dependent kinase 2 (cdk2) and cyclin E were down-regulated in HepG2-TR cells. Moreover, p21 protein or mRNA levels were up-regulated by around 5-fold or 7.3-fold respectively following T3 treatment. Furthermore, phospho-retinoblastoma (ppRb) protein was down-regulated by T3. The expression of TGF-β was studied to delineate the repression mechanism. TGF-β was stimulated by T3 and its promoter activity was enhanced six- to eight-fold by T3. Furthermore, both T3 and TGF-β repressed the expression of cdk2, cyclin E and ppRb. On the other hand, TGF-β neutralizing but not control antibody blocked the repression of cdk2, cyclin E and ppRb by T3. These results demonstrated that T3 might play a key role in liver tumor cell proliferation.

scholarly journals Identification of Novel Human Cdt1-binding Proteins by a Proteomics Approach: Proteolytic Regulation by APC/CCdh1

2008 ◽  
Vol 19 (3) ◽  
pp. 1007-1021 ◽  
Author(s):  
Nozomi Sugimoto ◽  
Issay Kitabayashi ◽  
Satoko Osano ◽  
Yasutoshi Tatsumi ◽  
Takashi Yugawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Binding Proteins ◽  
Mass Spectrometry Analysis ◽  
Anaphase Promoting Complex ◽  
Chromosomal Damage ◽  
Spectrometry Analysis

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it is central to the regulation of DNA replication during the cell cycle. In fact, unscheduled Cdt1 hyperfunction results in rereplication and/or chromosomal damage. Thus, it is important to understand its function and regulations precisely. We sought to comprehensively identify human Cdt1-binding proteins by a combination of Cdt1 affinity chromatography and liquid chromatography and tandem mass spectrometry analysis. Through this approach, we could newly identify 11 proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and IIα, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/CCdh1, SNF2H, topoisomerase I and IIα, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/CCdh1 indicated that this ubiquitin ligase controls the levels of Cdt1 during the cell cycle via three destruction boxes in the Cdt1 N-terminus. Notably, elimination of these destruction boxes resulted in induction of strong rereplication and chromosomal damage. Thus, in addition to SCFSkp2 and cullin4-based ubiquitin ligases, APC/CCdh1 is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.

Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1462-1462
Author(s):  
Michael Tallack ◽  
Thomas Whitington ◽  
Brooke Gardiner ◽  
Eleanor Wainwright ◽  
Janelle Keys ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Gene Promoters ◽  
Erythroid Cells ◽  
Red Cell Membrane ◽  
Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

Studies on Heme Oxygenase 1 During Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4254-4254
Author(s):  
Daniel Garcia Santos ◽  
Jesse Eisenberg ◽  
Matthias Schranzhofer ◽  
Prem Ponka
Keyword(s):  
Aminolevulinic Acid ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Erythroid Differentiation ◽  
The Body ◽  
Cellular Damage ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Erythroid Cells ◽  
Murine Erythroleukemia

Abstract Abstract 4254 Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures: No relevant conflicts of interest to declare.

TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4829-4829
Author(s):  
David C Dorn ◽  
Wei He ◽  
Joan Massague ◽  
Malcolm A.S. Moore
Keyword(s):  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Transforming Growth Factor Β ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

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