The role of cytokines and transcription factors on the regulation of megakaryocy topoiesis and platelet production are reviewed in this article. Megakaryocytopoiesis involves the proliferation and differentiation of megakaryocytic pro genitor cells into immature megakaryocytes, and the differentiation of immature megakaryocytes to mature megakaryocytes which produce platelets. The former is regulated mainly by thrombopoietin (TPO) and to a lesser degree by other cytokines such as interleukin-1 (IL-1), IL-3 and platelet-derived growth factor (PDGF), the later by TPO and probably IL-6 and IL-11. A number of transcription factors have been implicated in the control of megakaryocyte differentiation. GATA-1, FOG-1 and Fli-1 are essential regulators in early- and mid-stages of megakaryocytopoiesis. NF-E2 regulates late-stage of megakaryocytopoiesis and platelet production. However, the platelet release mechanism is poorly understood. Nitric oxide (NO) may act in the stage of platelet release through induction of apoptosis in megakaryocytes.
Animals ; Cytokines ; physiology ; DNA-Binding Proteins ; physiology ; Erythroid-Specific DNA-Binding Factors ; GATA1 Transcription Factor ; Hematopoiesis ; Humans ; Interleukins ; physiology ; Megakaryocytes ; physiology ; NF-E2 Transcription Factor ; NF-E2 Transcription Factor, p45 Subunit ; Platelet-Derived Growth Factor ; physiology ; Thrombopoiesis ; physiology ; Thrombopoietin ; physiology ; Transcription Factors ; physiology

OBJECTIVETo further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression.

METHODSBy transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2.

RESULTSIt was shown that when EDRF1 was overexpressed, production of alpha-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of alpha-, gamma-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression.

CONCLUSIONThe results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.

Cell Differentiation ; Cells, Cultured ; DNA-Binding Proteins ; genetics ; Erythroid-Specific DNA-Binding Factors ; Erythropoiesis ; physiology ; GATA1 Transcription Factor ; Gene Expression Regulation ; Globins ; genetics ; Humans ; NF-E2 Transcription Factor ; NF-E2 Transcription Factor, p45 Subunit ; RNA, Messenger ; analysis ; Transcription Factors ; genetics ; Up-Regulation

To observe the effects of Panax Notoginosides (PNS) on up-regulation of AP-1 family transcription factors NF-E2, c-jun and c-fos for exploring intracellular signal pathway of PNS in hematopoietic cells, four human hematopoietic cells lines including myeloid HL-60, erythroid K562, megakaryoid CHRF-288 and Meg-01 were incubated in the presence of PNS for 14 days. The nuclear protein of cells were extracted and analyzed by Western blot with antibodies against NF-E2, c-fos and c-jun. Electrophoretic mobility shift assay (EMSA) was performed by using (32)P labeled AP-1 consensus oligonucleotide which contains binding site for NF-E2, c-jun and c-fos. The results showed that the transcription factors NF-E2, c-jun and c-fos of AP-1 family could be induced by PNS. Western blot demonstrated that the nuclear protein of both NF-E2 and c-jun in four cell lines treated by PNS were increased by 1.5-2.5- and 2.0-3.0-fold over untreated cells respectively. The c-fos protein in three cell lines of K562, CHRF-288 and Meg-01 was also elevated by 2.0-3.0-fold respectively, while c-fos protein in HL-60 cells was no detectable difference after PNS treatment. EMSA results in four cell lines indicated that AP-1 binding activity initiated by PNS was apparently elevated to form higher density band of AP-1-DNA complex. In conclusion, the intracellular transcription regulation initiated by PNS was involved in transcription factors NF-E2, c-jun and c-fos of AP-1 family members, which could play an important role in the up-regulation of genes expression related to proliferation and differentiation of hematopoietic cells.
DNA ; metabolism ; DNA-Binding Proteins ; genetics ; Erythroid-Specific DNA-Binding Factors ; Gene Expression Regulation ; drug effects ; Genes, fos ; Genes, jun ; Ginsenosides ; pharmacology ; HL-60 Cells ; Humans ; K562 Cells ; NF-E2 Transcription Factor ; NF-E2 Transcription Factor, p45 Subunit ; Panax ; Transcription Factor AP-1 ; metabolism ; Transcription Factors ; genetics ; Up-Regulation

WT1 gene encodes a zinc finger transcription factor that regulates transcription of its downstream genes. Some of target genes for WT1 are involved in regulating both cell cycle and cellular proliferation and differentiation. However, WT1 itself is regulated by its upstream genes such as NF-kappaB and GATA-1. Thus there exists a pathway of transcriptional regulation mediated by WT1, which controls development of hematopoietic system. Leukemia results from disrupting the homeostasis among hematopoietic proliferation, differentiation and apoptosis, which is often the consequence of an inappropriate expression of transcription factors and subsequent disruption of the normal gene expression pattern. This article reviews the relationship between the WT1-mediated pathway of transcriptional regulation and leukemia.
Animals ; Carrier Proteins ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; DNA-Binding Proteins ; metabolism ; Erythroid-Specific DNA-Binding Factors ; GATA1 Transcription Factor ; Gene Expression Regulation ; Humans ; Leukemia ; etiology ; genetics ; NF-kappa B ; metabolism ; Nuclear Proteins ; genetics ; Retinoblastoma-Binding Protein 7 ; Transcription Factors ; metabolism ; Transcription, Genetic ; WT1 Proteins ; physiology

OBJECTIVETo investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2.

METHODSWe established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45.

RESULTSWe found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45.

CONCLUSIONNF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.

Base Sequence ; Blood Proteins ; genetics ; DNA Primers ; GATA1 Transcription Factor ; physiology ; Gene Expression Regulation ; physiology ; Gene Silencing ; HeLa Cells ; Humans ; Methylation ; Molecular Chaperones ; genetics ; NF-E2 Transcription Factor, p45 Subunit ; physiology ; Promoter Regions, Genetic ; Reverse Transcriptase Polymerase Chain Reaction

Mesenchymal stem cells (MSCs), precursors of diverse stromal cells, can support hematopoiesis in vitro and can promote the implantation of hematopoietic stem cells in vivo when co-transplanted with CD34(+) cells. The aim of this study was to investigate the potential effect of MSCs on the hematopoietic development of embryonic stem cells (ES cells) and the feasibility of a novel system in which ES cells will be co-cultured with MSCs. The murine bone marrow MSCs were isolated and cultured and then their phenotype and differentiation function were identified with FCM and histochemical technique. The CCE cells, murine ES cell line, were co-cultured with the isolated MSCs and the hematopoietic differentiation of CCE cells was observed with hematopoietic clonogenic assay and RT-PCR. The results showed that the morphology of MSCs became gradually homogeneous with the passage culture of cells. After passage 4, the marker of Sca-1, CD29, CD44 and CD105 were highly expressed, however, CD34 and CD45, the specific marker of hematopoietic and endothelial cells, could hardly be identified. The isolated MSCs differentiated into adipocytes and osteoblasts in specific induction culture system. After maintaining culture on mouse embryonic fibroblasts, CCE cells were plated in suspended culture system with only differentiation inductive agents and co-culture system in which MSCs were added. Compared with CCE cell suspended culture, the cells differentiated into embryoid body were obviously enhanced and there were no colony-forming cells in the co-culture system of ES cells and MSCs. In addition, transcription factor Oct-4 in co-cultured CCE cells was expressed and hematopoietic markers, Flk-1, GATA-1 and beta-H1, were negative. The ability of embryoid bodies derived from the co-culture system to produce hematopoietic colonies was markedly higher than that from the suspended culture system. It is concluded that MSCs inhibit the initial differentiation of ESC and enhance hematopoietic differentiation ability of the co-cultured ES cells.
Animals ; Bone Marrow Cells ; physiology ; Cell Differentiation ; Coculture Techniques ; DNA-Binding Proteins ; genetics ; Embryo, Mammalian ; cytology ; Erythroid-Specific DNA-Binding Factors ; Female ; GATA1 Transcription Factor ; Gene Expression ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mesenchymal Stromal Cells ; physiology ; Mice ; Mice, Inbred C57BL ; Octamer Transcription Factor-3 ; Transcription Factors ; genetics

Embryonic hematopoiesis in mammals is characterized by successive temporal and spatial changes. Previous investigations indicate that in vitro differentiation of embryonic stem cells (ES cells) derived from 129 mice can mimic embryonic hematopoiesis to some extent. To investigate the in vitro hematopoietic differentiation capacity of ES cells derived from C57BL/6 mice, the authors initially established the murine ES cell line with standard identification methods employed. Next, two-step culture system was utilized for embryoid bodies formation and the appearance of different hematopoietic precursors was confirmed by CFC assay, cellular chemical staining as well as RT-PCR. The results demonstrated that the ES cell line MES-1 fulfilled the criteria of ES cell line and its progeny after in vitro differentiation included primitive and definitive erythrocyte precursors, mixed colony-forming cells and granulocyte/macrophage colony-forming cells. RT-PCR analysis revealed the molecular consistence of transcription factors and hematopoietic markers with cellular event. In conclusion, MES-1 established from C57BL/6 mice was able to differentiate in vitro to a variety of hematopoietic precursors, thus could partly recapitulate embryonic hematopoiesis.
Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; genetics ; Cell Line ; Colony-Forming Units Assay ; DNA-Binding Proteins ; genetics ; Embryo, Mammalian ; cytology ; Erythroblasts ; cytology ; metabolism ; Erythroid Precursor Cells ; cytology ; metabolism ; Erythroid-Specific DNA-Binding Factors ; Gene Expression ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; Time Factors ; Transcription Factors ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics

OBJECTIVETo explore the mechanism of acupuncture in delaying aging.

METHODSUsing SAMP10 mice and normal control SAMR1 as model and applying RT-PCR and DIG probed Northern blot techniques to observe expression of NF-E2, YB-1, LRG47 genes in whole brain, cortex and hippocampus in the 8-month SAMR1 control group, 8-month SAMP10 control group, 8-month SAMP10 acupuncture group and 8-month SAMP10 non-point acupuncture group.

RESULTSIn the SAMP10 control group, the expression of NF-E2, YB-1 and LRG47 were down-regulated in the whole brain, cortex and hippocampus, and after acupuncture they were up-regulated and tended to normal.

CONCLUSIONAging of the SAMP10 mouse brain is related with expression of NF-E2, YB-1 and LRG47 genes, and acupuncture can regulate the expression of NF-E2, YB-1 and LRG47 genes, improving the functions of erythrocyte series, increasing proliferation of cells and immune function of cells in anti-bacteria, hence anti-aging.

Acupuncture Therapy ; Aging ; metabolism ; Animals ; Brain ; metabolism ; Female ; GTP-Binding Proteins ; genetics ; Gene Expression Regulation ; Male ; Mice ; NF-E2 Transcription Factor, p45 Subunit ; genetics ; RNA, Messenger ; analysis ; Y-Box-Binding Protein 1 ; genetics

OBJECTIVETo find out how GATA-1 and GATA-2 behave in the bone marrow of patients with Monge's disease.

METHODSThe levels of mRNA in mononuclear cells (MNC) and proteins of GATA-1 and GATA-2 in the bone marrow of patients with Monge's disease and controls were determined by RT-PCR and immune cytolysis chemical method.

RESULTS(1) All patients and controls expressed GATA-1 mRNA (Monge's disease 1.033 +/- 0.146, Control 0.458 +/- 0.076) and GATA-2 mRNA (Monge's disease 0.451 +/- 0.073, Control 0.185 +/- 0.074). All patients expressed both GATA-1 (positive cell counts 77.3 +/- 33.3, positive score 135.4 +/- 75.4) and GATA-2 ( positive cell counts 29.4 +/- 11.4, positive score 48.4 +/- 19.7). All the controls expressed GATA-1 (positive cell counts 18.1 +/- 11.3, positive score 24.2 +/- 13.4) while 12 of 20 controls expressed GATA-2 ( positive cell counts 5.4 +/- 3.0, positive score 7.3 +/- 4.2). The expression of mRNA and proteins of GATA-1 and GATA-2 in Monge's disease were higher than in controls (P < 0.01). (2) There was a positive correlation between GATA-1 and Hb (P < 0.01), as did between mRNA and proteins of GATA-1 and GATA-2. (3) Both the proteins of GATA-1 and GATA-2 located only in the cytoplasm but not the nucleus.

CONCLUSIONSTwo of inherent genes, GATA-1 and GATA-2 which were expressed at higher levels in patients with Monge's disease than in controls might play significant roles in the pathogenesis of Monge's disease.

Adult ; Altitude Sickness ; metabolism ; GATA1 Transcription Factor ; metabolism ; GATA2 Transcription Factor ; metabolism ; Humans ; Male ; Polycythemia ; metabolism ; RNA, Messenger ; metabolism

OBJECTIVETo investigate the effect of simulated microgravity on erythroid differentiation of K562 cells and explore the possible mechanism.

METHODSThe fourth generation rotating cell culture system was used to generate the simulated microgravity environment. Benzidine staining was used to evaluate the cell inhibition rate, and real-time quantitative PCR (qRT-PCR) was used to detect GATA-1, GATA-2, Ets-1, F-actin, β-Tubulin and vimentin mRNA expressions. The changes of cytoskeleton were observed by fluorescence microscopy, and Western blotting was employed to assay F-actin, β-tubulin and vimentin protein expression levels.

RESULTSBenzidine staining showed that simulated microgravity inhibited erythroid differentiation of K562 cells. K562 cells treated with Hemin presented with increased mRNA expression of GATA-1 and reduced GATA-2 and Ets-1 mRNA expressions. Simulated microgravity treatment of the cells resulted in down-regulated GATA-1, F-actin, β-tubulin and vimentin mRNA expressions and up-regulated mRNA expressions of GATA-2 and Ets-1, and reduced F-actin, β-tubulin and vimentin protein expressions. Exposure to simulated microgravity caused decreased fluorescence intensities of cytoskeletal filament F-actin, β-tubulin and vimentin in the cells.

CONCLUSIONSimulated microgravity inhibits erythroid differentiation of K562 cells possibly by causing cytoskeleton damages to result in down-regulation of GATA-1 and up-regulation of GATA-2 and Ets-1 expressions.

Actins ; metabolism ; Cell Differentiation ; Down-Regulation ; GATA1 Transcription Factor ; metabolism ; GATA2 Transcription Factor ; metabolism ; Hemin ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Protein c-ets-1 ; metabolism ; Tubulin ; metabolism ; Up-Regulation ; Vimentin ; metabolism ; Weightlessness Simulation