The Effects And Mechanisms Of Microgravity Simulated On Erythroid Differentiation Of K562Cells

BackgroundWith the aerospace industry development, human development to the field of space and competition is more intense. In2005, the succession of launching "Shen Zhou Ⅴ" manned spacecraft opened a new chapter in China Manned Space Engineering, in2008, the "Shen Zhou Ⅶ" manned spacecraft was successfully launched, and our astronauts achieved the first space extravehicular activities. With the prosperity and progress in the aviation industry, it has become increasingly concerned about the effect of space flight on astronauts at the same time, and the primary issue of space hematology is red blood cell declining in the quality. The early space medical scientists found that for1960-1990in the United States and the former Soviet Union astronaut researches, changing of the blood system is expressed as the total hemoglobin, red blood cell count and red blood cell volume decreased and the number of reticulocytes increased. They named it "spaceflight anemia". And the hematological changes become more obvious with the extension of flight time. This is not only hindered to completion of their space mission but also caused potential hazards to blood system. So, causes and prevention measures of "spaceflight anemia" appear to become the research focus of the current space hematology.Simulated microgravity and erythroid differentiation Some studies show that simulated microgravity can inhibit the proliferation of UT7/EPO cells, the reason may simulated microgravity can active the caspases-3and down regulate the expression of Bcl-XL protein, in our study, we fund that, simulated microgravity can effect the phosphorylation of ERK1/2protein, and cell cycle stop at G0/G1phase, and then, inhibit cell proliferation. Under simulated microgravity, the expression of EPOR on K562cells declined, and in our laboratory, simulated microgravity can affect the expression of GATA-1mRNA in cord blood sorted CD34+cells and inhibit the erythroid differentiation. The migratory capacity of reduced of CD34+cells after cultured in RCCS2or3days, the main performance is the chemotactic response of SDF-1a decreased, and related to the chemotactic effect of SDF-1a cytoskeleton also changed, the expression of F-actin cytoskeleton down while migratory capacity of lymphocytes and osteoblasts was inhibited and accompanied by changes in the cytoskeleton, this may be one reason of reduced of red blood cell count. In summary, simulated microgravity through a variety of ways to affect the proliferation and differentiation of erythroid and lead to the symptoms of "anemia" and the study of specific mechanisms is a hot issue in the field.The process of generation of various types of blood cells by hematopoietic stem cell differentiation to the progenitor cells for multiple are proliferation to the gradual differentiation of erythroid progenitor cells, erythroid precursor cells, and finally develop into the physiological functions of mature cells. Under normal ground environment, the number of red blood cells maintained by the generation and apoptosis. A stable hematopoietic microenvironment is very important for maintaining a balance between the various stages of erythroid proliferation, differentiation, growth and apoptosis. Hematopoietic microenvironment consists of microvascular system, nerves, reticular cells, extracellular matrix and stromal cells and it secreted cytokines. And the role of cytokines involved in hematopoietic regulation can be divided into the positive control and negative regulation. The positive controls include stem cell factor (SCF), erythropoietin (EPO), thrombopoietin (TPO), colony-stimulating factor (G-CSF and GM-CSF) and most of interleukin. The negative regulations involve interferon (IFN), tumor necrosis factor (TNF), transforming growth factor beta (TGF-β), erythropoietin (EPO), a member of the positive control, plays an important role in red blood cell survival, proliferation, differentiated and red blood cell mature.Erythropoietin (EPO) is an erythroid stem cells in hematopoietic factor witch produced by the kidneys, and generate in the absence of oxygen. It through prevents apoptosis balance to promote proliferation of erythroid progenitor cells, and activates several signaling pathways involved in erythroid differentiation into mature red blood cells. It’s a glycoprotein hormone, which can combine with EPO receptor (EPOR), and recruit of EPOR monomer to forming a dimmer, and phosphorylation JAK tyrosine kinase, activate multiple signaling pathways that playing a biological effect. Researches have been shown that, mice that absenting of EPO or knocking with EPOR gene would be died of severe anemia in12.5days of embryonic, mouse with the liver cells in absence of EPO and EPOR knockout, red blood cell colony-forming units (CFU-E) and burst erythroid colony-forming units (BFU-E) as the same with the number of normal cells, but can not further differentiate into red blood cells containing hemoglobin. The role of EPO and EPOR may be promoting erythroid differentiation and maturation in differentiation of erythroid progenitor cells in the late.EPOR belongs to the type I single transmembrane cytokine receptor super family, relative molecular mass is62×103, consists of507amino acids, including extracellular, transmembrane and cytoplasmic. The families have a conservative extracellular aptamers area, they are connected by fiber proteins Ⅲ like region and a conserved β chains. The extracellular region of each monomer contains two FNIII-like sub domains (D1and D2) each of which utilizes seven b-strands to form two anti-parallel b-pleated sheets. However, EPO binding to its receptor induces the activation of the receptor-associated JAK2tyrosine kinase and stimulates tyrosine phosphorylation of the receptor itself and other proteins, Subsequently, several intracellular signaling pathways are activated, including mitogen-activated protein kinases, STAT5, SHIP-1, SHC, Vav, Gab1, Gab2, SYK, SHIP, IRS-2and EPOR, Activation of several signaling pathways Participate in the proliferation and differentiation of erythroid.3-phosphoinositide kinase (PI3K) is a widely presents in most tissue cells of the human kinase and also exists in the human erythroleukemia cell line K562cell membrane. The class IA family of PI3-kinases consists of a regulatory subunit (p85) and a catalytic subunit (p110), and composition with three different genes pik3rl, pik3r2and pik3r3. it encodes at least seven kinds of adapter protein or mediation protein, P110catalytic subunit of three subtypes have been identified, they are α、β and y witch encoded by three different genes in PI3KCA, PI3KCB and PI3KCD, It has been shown that the p85regulatory subunit of PtdIns3-Kassociates, via its Src homology2domains, with the tyrosine-phosphorylated72-kDa form of the EPO receptor within minutes of EPO binding at the plasma membrane level, This combination is considered as the start of the regulation of erythroid progenitor cell proliferation, differentiation and maturation. Activated PI3K can produce two kinds of liposomes (PI-3,4-P2and PI-3,4,5-P3) that activate AKT as a second messenger. AKT is a serine/threonine protein kinase, also known as protein kinase B witch has a wide range of expression in mammals. AKT has3subtypes, AKT1/alpha, AKT2/beta, and AKT3/y respectively, they have a high degree of similarity, more than80%of the homologous sequences although by a different gene expression, AKT1has an extensive and high level of expression in the liver, spleen, kidney, AKT2is expressed in skeletal muscle, intestine and reproductive organs, AKT3unlike AKT1and AKT2with a wide range of expression in the body, it only expression in brain tissue and testicular. In K562cells, AKT mainly express in the cytoplasm. AKT through its NH2structure of regional combined with phosphorylation of PI3K liposomes photogenic in the cell membrane, and then AKT threonine308phosphorylation (through PDK1), and it’s Serine473phosphorylation by EPO, and AKT be completely active. Activated AKT nuclear translocation from the cytoplasm into the nucleus to participate in signal transduction, AKT-mediated signaling pathways involved in cell survival, proliferation and anti-apoptotic, Erythropoietin stimulates phosphorylation and activation of GATA-1via the PI3-kinase/AKT signaling pathway.GATA-1is an important transcription factor in late expression of the erythroid progenitor cell differentiation, it belongs to the zinc finger transcription factor family, the family includes several members, GATA-1, GATA-2and GATA-3is the basis of hematopoietic growth factors; GATA-4, GATA-5and GATA-6express in heart, lung and intestinal cells. GATA-1expression in red blood cells, mast cells of the nucleus in megakaryocytic, GATA-2can activate the endogenous expression of GATA-1, GATA-1involved in transcriptional regulation in three ways:binding to the promoter complex composed, Enhance promoter activity through binds to the enhancer, act on the globin gene promoter and enhancer in erythroid cells and a, β-globin locus control region, GATA-1in the early stages of erythroid progenitor cells could significantly promote the proliferation of erythroid, and it an important factor in differentiation, maturation indispensable in the late stage of erythroid progenitor cells. In the late erythroid differentiation, GATA-1expression levels will decline, This may be due to ultimately generate mature red blood cells in the early stages of erythroid differentiation of GATA-1expression is sufficient to maintain the amount had not generated, So along with the continuous differentiation of erythroid GATA-1continue to be consumed, and its expression level gradually decreased. GATA-1can promote the expression of endogenous GATA-1, EKLF, beta globin and est.-1gene, etc, and endogenous EPOR expression also elevated. Those erythroid gene expression levels related with the EPO signal pathway, some studies showed that PI3K/AKT is the an important pathway in expression of GATA-1in the EPO-induced K562cells to erythroid differentiation and mature, under the effect of EPO, the GATA-1S310residues can be phosphorylated and to be activated, activated GATA-1has biological functions, Gene sequences can be found in almost all the red lines expressing the gene encoding the globin regulatory regions of GATA-1binding, GATA-1can also play an anti-apoptotic effects by regulating the expression of Bcl-XL, whether Simulated microgravity affect PI3K/AKT pathway downstream of GATA-1and erythroid proliferation, differentiation-related protein expression in erythroid differentiation have not been reported.ObjectWhether simulated microgravity affect EPO induced erthroid differentiation via affect the PI3K/AKT pathway and its downstream expression of GATA-1that related to erythroid proliferation, differentiation expression in K562cells?MethodsIn this study, the rotary culture system (RCCS4) simulated microgravity, established K562cell erythroid as an differentiation model, study the simulated microgravity on erythroid differentiation of K562cells and its mechanism, The detail of technical route:first, K562cells were induced to erythroid differentiation by rhEPO, second, Flow cytometry to detect K562cell surface expression of EPO receptor and CD71, Expression levels of GATA-1, EPOR and beta-globin in K562cells by RT-PCR, third, AKT and GATA-1expression in K562cells detected by western-blot analysis method, Preliminary exploration of the mechanism of simulated microgravity affecting on K562cells to erythroid differentiation.Results and conclusionOur study found that simulated microgravity can inhibit the rhEPO-induced K562cells to erythroid differentiation, the expression of CD71and EPOR decreased, and Induction of apoptosis, Simulated microgravity inhibits the expression level of mRNA expression of GATA-1, EPOR in beta-globin in K562cells, PI3K/AKT pathway mediated erythroid differentiation protein AKT and its downstream GATA-1protein levels in the expression decreased under simulated microgravity. These results may be put forward some new ideas for the study of "spaceflight anemia".