J2E erythrocytic differentiation (EPO): Difference between revisions
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|TCOverview=Erythropoietin (Epo) is the hormone, which regulates red blood cell production1. It is produced primarily in the kidney, and binds to Epo receptors (Epor) on the surface of immature erythroid cells in the bone marrow, thereby initiating the final stages of red cell maturation[1,2]. Following binding of Epo to its cognate receptor, a series of intracellular signaling cascades are activated, including stimulation of the JAK/STAT and ras/MAP kinase pathways[3,4]. This leads to enhanced cell division, followed by terminal differentiation which is characterized by the production of hemoglobin. In addition, morphological changes occur involving a reduction in cell size, nuclear condensation, and eventually extrusion of the nucleus to produce reticulocytes. Mature red blood cells (erythrocytes) containing large amounts of hemoglobin then circulate around the body transporting oxygen and carbon dioxide [5].<br><br>References:<br>[1] Bunn HF. Erythropoietin. Cold Spring Harbor perspectives in medicine 2013; 3: a011619.<br>[2] Koury MJ, Koury ST, Bondurant MC, Graber SE. Correlation of the molecular and anatomical aspects of renal erythropoietin production. Contributions to nephrology 1989; 76: 24-29; discussion 30-22.<br>[3] Richmond TD, Chohan M, Barber DL. Turning cells red: signal transduction mediated by erythropoietin. Trends Cell Biol 2005; 15: 146-155.<br>[4] Ingley E. Integrating novel signaling pathways involved in erythropoiesis. IUBMB Life 2012; 64: 402-410.<br>[5] Palis J. Primitive and definitive erythropoiesis in mammals. Frontiers in physiology 2014; 5: 3.<br> | |TCOverview=Erythropoietin (Epo) is the hormone, which regulates red blood cell production1. It is produced primarily in the kidney, and binds to Epo receptors (Epor) on the surface of immature erythroid cells in the bone marrow, thereby initiating the final stages of red cell maturation[1,2]. Following binding of Epo to its cognate receptor, a series of intracellular signaling cascades are activated, including stimulation of the JAK/STAT and ras/MAP kinase pathways[3,4]. This leads to enhanced cell division, followed by terminal differentiation which is characterized by the production of hemoglobin. In addition, morphological changes occur involving a reduction in cell size, nuclear condensation, and eventually extrusion of the nucleus to produce reticulocytes. Mature red blood cells (erythrocytes) containing large amounts of hemoglobin then circulate around the body transporting oxygen and carbon dioxide [5].<br><br>References:<br>[1] Bunn HF. Erythropoietin. Cold Spring Harbor perspectives in medicine 2013; 3: a011619.<br>[2] Koury MJ, Koury ST, Bondurant MC, Graber SE. Correlation of the molecular and anatomical aspects of renal erythropoietin production. Contributions to nephrology 1989; 76: 24-29; discussion 30-22.<br>[3] Richmond TD, Chohan M, Barber DL. Turning cells red: signal transduction mediated by erythropoietin. Trends Cell Biol 2005; 15: 146-155.<br>[4] Ingley E. Integrating novel signaling pathways involved in erythropoiesis. IUBMB Life 2012; 64: 402-410.<br>[5] Palis J. Primitive and definitive erythropoiesis in mammals. Frontiers in physiology 2014; 5: 3.<br> | ||
|TCQuality_control=Expression of the following genes was assessed to determine the validity of this cell line as a model of Epo-induced erythroid differentiation (Figure 2). All these genes are required for normal erythroid differentiation.<br><br>* Epor mediates epo-induced proliferation and differentiation [10]<br>* Alas2 is the rate limiting enzyme for the Heme biosynthesis pathway [11]<br>* Hbb-b1 hemoglobin, adult beta major chain required for oxygen transport [12]<br>* Gata-1 is an essential transcription factor for erythroid development [13, 14]<br>* Klf1 is a key transcriptional regulator for erythroid development [15]<br>* Nfe2 regulates erythroid maturation [16]<br><br><html><img src=' | |TCQuality_control=Expression of the following genes was assessed to determine the validity of this cell line as a model of Epo-induced erythroid differentiation (Figure 2). All these genes are required for normal erythroid differentiation.<br><br>* Epor mediates epo-induced proliferation and differentiation [10]<br>* Alas2 is the rate limiting enzyme for the Heme biosynthesis pathway [11]<br>* Hbb-b1 hemoglobin, adult beta major chain required for oxygen transport [12]<br>* Gata-1 is an essential transcription factor for erythroid development [13, 14]<br>* Klf1 is a key transcriptional regulator for erythroid development [15]<br>* Nfe2 regulates erythroid maturation [16]<br><br><html><img src='/resource_browser/images/TC_qc/J2E_Fig2.png' onclick='javascript:window.open("/resource_browser/images/TC_qc/J2E_Fig2.png", "imgwindow", "width=768,height=785");' style='width:700px;cursor:pointer'/></html>Figure 2. Expression of key genes associated with erythroid differentiation. TPM: Tags per million.<br><br>Refereneces:<br>[6] Klinken SP, Nicola NA, Johnson GR. In vitro-derived leukemic erythroid cell lines induced by a raf- and myc-containing retrovirus differentiate in response to erythropoietin. Proc Natl Acad Sci U S A 1988; 85: 8506-8510.<br>[7] Tilbrook PA, Bittorf T, Callus BA, Busfield SJ, Ingley E, Klinken SP. Regulation of the erythropoietin receptor and involvement of JAK2 in differentiation of J2E erythroid cells. Cell Growth Differ 1996; 7: 511-520.<br>[8] Tilbrook PA, Ingley E, Williams JH, Hibbs ML, Klinken SP. Lyn tyrosine kinase is essential for erythropoietin-induced differentiation of J2E erythroid cells. EMBO J 1997; 16: 1610-1619.<br>[9] Busfield SJ, Klinken SP. Erythropoietin-induced stimulation of differentiation and proliferation in J2E cells is not mimicked by chemical induction. Blood 1992; 80: 412-419.<br>[10] Lodish HF, Hilton DJ, Klingmuller U, Watowich SS, Wu H. The erythropoietin receptor: biogenesis, dimerization, and intracellular signal transduction. Cold Spring Harb Symp Quant Biol 1995; 60: 93-104.<br>[11] Meguro K, Igarashi K, Yamamoto M, Fujita H, Sassa S. The role of the erythroid-specific delta-aminolevulinate synthase gene expression in erythroid heme synthesis. Blood 1995; 86: 940-948.<br>[12] Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation. Exp Hematol 2005; 33: 259-271.<br>[13] Tsai SF, Martin DI, Zon LI, D'Andrea AD, Wong GG, Orkin SH. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature 1989; 339: 446-451.<br>[14] Whitelaw E, Tsai SF, Hogben P, Orkin SH. Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse. Mol Cell Biol 1990; 10: 6596-6606.<br>[15] Miller IJ, Bieker JJ. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Mol Cell Biol 1993; 13: 2776-2786.<br>[16] Andrews NC, Erdjument-Bromage H, Davidson MB, Tempst P, Orkin SH. Erythroid transcription factor NF-E2 is a haematopoietic-specific basic- leucine zipper protein. Nature 1993; 362: 722-728.<br> | ||
|TCSample_description='''J2E model of Erythocytic differentiation'''<br><br>J2E cells are murine fetal liver cells that have been immortalised with the J2 retrovirus. J2E cells retain the capacity to respond to Epo by terminally differentiating and synthesizing hemoglobin6. The mouse J2E cell line responds to Epo by activating the JAK/STAT and ras/MAP kinase pathways7, as well as a novel Lyn-signaling cascade that we identified8. As a consequence of exposure to Epo, the cells undergo a burst of proliferation, followed by entry into the terminally differentiated state by synthesizing hemoglobin and changing morphologically9. These cells, therefore, provide a very good model for normal erythroid maturation in response to Epo.<br>J2E cells are maintained in DMEM (Gibco) 5% FCS (Bovogen Biologicals) at 370C and 5% CO2. Cell density is kept at 5-8 X105 cells/ml. Cells were induced with 5U/ml of Epo (Eprex®) (Jannsen). At least 1 X 107 cells were collected for RNA at 0min, 15min, 30min, 45min,1h, 1h 20min, 1h 40min, 2h, 2h 30min,3h, 3h 30min, 4h, 6h, 12h, 24h and 48h.<br><br>'''Key marker for differentiation'''<br>Enumeration of benzidine positive cells, as an indication of hemoglobin synthesis, was carried out to monitor differentiation. The time course of Epo-induced differentiation of J2E cells shows that hemoglobin production increases markedly 24-48h after stimulation (Figure 1).<br><br><html><img src=' | |TCSample_description='''J2E model of Erythocytic differentiation'''<br><br>J2E cells are murine fetal liver cells that have been immortalised with the J2 retrovirus. J2E cells retain the capacity to respond to Epo by terminally differentiating and synthesizing hemoglobin6. The mouse J2E cell line responds to Epo by activating the JAK/STAT and ras/MAP kinase pathways7, as well as a novel Lyn-signaling cascade that we identified8. As a consequence of exposure to Epo, the cells undergo a burst of proliferation, followed by entry into the terminally differentiated state by synthesizing hemoglobin and changing morphologically9. These cells, therefore, provide a very good model for normal erythroid maturation in response to Epo.<br>J2E cells are maintained in DMEM (Gibco) 5% FCS (Bovogen Biologicals) at 370C and 5% CO2. Cell density is kept at 5-8 X105 cells/ml. Cells were induced with 5U/ml of Epo (Eprex®) (Jannsen). At least 1 X 107 cells were collected for RNA at 0min, 15min, 30min, 45min,1h, 1h 20min, 1h 40min, 2h, 2h 30min,3h, 3h 30min, 4h, 6h, 12h, 24h and 48h.<br><br>'''Key marker for differentiation'''<br>Enumeration of benzidine positive cells, as an indication of hemoglobin synthesis, was carried out to monitor differentiation. The time course of Epo-induced differentiation of J2E cells shows that hemoglobin production increases markedly 24-48h after stimulation (Figure 1).<br><br><html><img src='/resource_browser/images/TC_qc/J2E_Fig1.png'></html><br>Figure 1. Benzidine positive cells were enumerated at each time point. Three biological replicates were analysed.<br> | ||
|Time_Course= | |Time_Course= | ||
|category_treatment= | |category_treatment=Differentiation | ||
|collaborators=Peter Klinken | |collaborators=Peter Klinken | ||
|description=mouse_J2E | |description=mouse_J2E | ||
| Line 14: | Line 14: | ||
|series=IN_VITRO DIFFERENTIATION SERIES | |series=IN_VITRO DIFFERENTIATION SERIES | ||
|species=Mouse (Mus musculus) | |species=Mouse (Mus musculus) | ||
|tet_config= | |tet_config=https://fantom.gsc.riken.jp/5/suppl/tet/EPO.tsv.gz | ||
|tet_file=https://fantom.gsc.riken.jp/5/tet#!/search/?filename=mm9.cage_peak_phase1and2combined_tpm_ann_decoded.osc.txt.gz&file=1&c=1&c=134&c=131&c=132&c=135&c=136&c=137&c=138&c=141&c=142&c=143&c=144&c=145&c=146&c=147&c=148&c=149&c=150&c=151&c=152&c=153&c=157&c=154&c=155&c=158&c=159&c=160&c=161&c=162&c=163&c=164&c=165&c=166&c=167&c=168&c=169&c=170&c=171&c=172&c=173&c=176&c=175&c=177&c=178&c=179&c=180&c=181&c=182 | |||
|time_points= | |time_points= | ||
|time_span=48 hours | |time_span=48 hours | ||
|timepoint_design= | |timepoint_design=Early focus | ||
|tissue_cell_type= | |tissue_cell_type=Erythrocytes | ||
|zenbu_config= | |zenbu_config=https://fantom.gsc.riken.jp/zenbu/gLyphs/#config=S2tJQUUwFZVqKAAH5_L8qC | ||
}} | }} | ||
Latest revision as of 17:29, 14 March 2022
| Series: | IN_VITRO DIFFERENTIATION SERIES |
|---|---|
| Species: | Mouse (Mus musculus) |
| Genomic View: | Zenbu |
| Expression table: | FILE |
| Link to TET: | TET |
| Sample providers : | Peter Klinken |
| Germ layer: | mesoderm |
| Primary cells or cell line: | cell line |
| Time span: | 48 hours |
| Number of time points: | 16 |
| Overview |
|---|
|
Erythropoietin (Epo) is the hormone, which regulates red blood cell production1. It is produced primarily in the kidney, and binds to Epo receptors (Epor) on the surface of immature erythroid cells in the bone marrow, thereby initiating the final stages of red cell maturation[1,2]. Following binding of Epo to its cognate receptor, a series of intracellular signaling cascades are activated, including stimulation of the JAK/STAT and ras/MAP kinase pathways[3,4]. This leads to enhanced cell division, followed by terminal differentiation which is characterized by the production of hemoglobin. In addition, morphological changes occur involving a reduction in cell size, nuclear condensation, and eventually extrusion of the nucleus to produce reticulocytes. Mature red blood cells (erythrocytes) containing large amounts of hemoglobin then circulate around the body transporting oxygen and carbon dioxide [5]. |
| Sample description |
|---|
|
J2E model of Erythocytic differentiation |
| Quality control |
|---|
|
Expression of the following genes was assessed to determine the validity of this cell line as a model of Epo-induced erythroid differentiation (Figure 2). All these genes are required for normal erythroid differentiation. |
Figure 2. Expression of key genes associated with erythroid differentiation. TPM: Tags per million.Profiled time course samples
Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples
| 13063-139I3 | J2E erythroblastic leukemia response to erythropoietin | 00hr00min | biol_rep1 |
| 13064-139I4 | J2E erythroblastic leukemia response to erythropoietin | 00hr15min | biol_rep1 |
| 13065-139I5 | J2E erythroblastic leukemia response to erythropoietin | 00hr30min | biol_rep1 |
| 13066-139I6 | J2E erythroblastic leukemia response to erythropoietin | 00hr45min | biol_rep1 |
| 13067-139I7 | J2E erythroblastic leukemia response to erythropoietin | 01hr00min | biol_rep1 |
| 13068-139I8 | J2E erythroblastic leukemia response to erythropoietin | 01hr20min | biol_rep1 |
| 13069-139I9 | J2E erythroblastic leukemia response to erythropoietin | 01hr40min | biol_rep1 |
| 13070-140A1 | J2E erythroblastic leukemia response to erythropoietin | 02hr00min | biol_rep1 |
| 13071-140A2 | J2E erythroblastic leukemia response to erythropoietin | 02hr30min | biol_rep1 |
| 13072-140A3 | J2E erythroblastic leukemia response to erythropoietin | 03hr00min | biol_rep1 |
| 13073-140A4 | J2E erythroblastic leukemia response to erythropoietin | 03hr30min | biol_rep1 |
| 13074-140A5 | J2E erythroblastic leukemia response to erythropoietin | 04hr | biol_rep1 |
| 13075-140A6 | J2E erythroblastic leukemia response to erythropoietin | 06hr | biol_rep1 |
| 13076-140A7 | J2E erythroblastic leukemia response to erythropoietin | 12hr | biol_rep1 |
| 13077-140A8 | J2E erythroblastic leukemia response to erythropoietin | 24hr | biol_rep1 |
| 13078-140A9 | J2E erythroblastic leukemia response to erythropoietin | 48hr | biol_rep1 |
| 13129-140G6 | J2E erythroblastic leukemia response to erythropoietin | 00hr00min | biol_rep2 |
| 13130-140G7 | J2E erythroblastic leukemia response to erythropoietin | 00hr15min | biol_rep2 |
| 13132-140G9 | J2E erythroblastic leukemia response to erythropoietin | 00hr45min | biol_rep2 |
| 13133-140H1 | J2E erythroblastic leukemia response to erythropoietin | 01hr00min | biol_rep2 |
| 13134-140H2 | J2E erythroblastic leukemia response to erythropoietin | 01hr20min | biol_rep2 |
| 13135-140H3 | J2E erythroblastic leukemia response to erythropoietin | 01hr40min | biol_rep2 |
| 13136-140H4 | J2E erythroblastic leukemia response to erythropoietin | 02hr00min | biol_rep2 |
| 13137-140H5 | J2E erythroblastic leukemia response to erythropoietin | 02hr30min | biol_rep2 |
| 13138-140H6 | J2E erythroblastic leukemia response to erythropoietin | 03hr00min | biol_rep2 |
| 13139-140H7 | J2E erythroblastic leukemia response to erythropoietin | 03hr30min | biol_rep2 |
| 13140-140H8 | J2E erythroblastic leukemia response to erythropoietin | 04hr | biol_rep2 |
| 13141-140H9 | J2E erythroblastic leukemia response to erythropoietin | 06hr | biol_rep2 |
| 13142-140I1 | J2E erythroblastic leukemia response to erythropoietin | 12hr | biol_rep2 |
| 13143-140I2 | J2E erythroblastic leukemia response to erythropoietin | 24hr | biol_rep2 |
| 13144-140I3 | J2E erythroblastic leukemia response to erythropoietin | 48hr | biol_rep2 |
| 13195-141E9 | J2E erythroblastic leukemia response to erythropoietin | 00hr00min | biol rep3 |
| 13196-141F1 | J2E erythroblastic leukemia response to erythropoietin | 00hr15min | biol_rep3 |
| 13197-141F2 | J2E erythroblastic leukemia response to erythropoietin | 00hr30min | biol_rep3 |
| 13198-141F3 | J2E erythroblastic leukemia response to erythropoietin | 00hr45min | biol_rep3 |
| 13199-141F4 | J2E erythroblastic leukemia response to erythropoietin | 01hr00min | biol_rep3 |
| 13200-141F5 | J2E erythroblastic leukemia response to erythropoietin | 01hr20min | biol_rep3 |
| 13201-141F6 | J2E erythroblastic leukemia response to erythropoietin | 01hr40min | biol_rep3 |
| 13202-141F7 | J2E erythroblastic leukemia response to erythropoietin | 02hr00min | biol rep3 |
| 13203-141F8 | J2E erythroblastic leukemia response to erythropoietin | 02hr30min | biol_rep3 |
| 13204-141F9 | J2E erythroblastic leukemia response to erythropoietin | 03hr00min | biol_rep3 |
| 13205-141G1 | J2E erythroblastic leukemia response to erythropoietin | 03hr30min | biol_rep3 |
| 13206-141G2 | J2E erythroblastic leukemia response to erythropoietin | 04hr | biol_rep3 |
| 13207-141G3 | J2E erythroblastic leukemia response to erythropoietin | 06hr | biol_rep3 |
| 13208-141G4 | J2E erythroblastic leukemia response to erythropoietin | 12hr | biol_rep3 |
| 13209-141G5 | J2E erythroblastic leukemia response to erythropoietin | 24hr | biol_rep3 |
| 13210-141G6 | J2E erythroblastic leukemia response to erythropoietin | 48hr | biol_rep3 |
