GPS2 Promotes Erythropoiesis By Stabilizing EKLF

Erythropoiesis is a complex multistep process that begins from pluripotent hematopoietic stem cells?HSCs?and terminates with the production of functional erythrocytes?RBCs?and involves many key cellular events including lineage commitment,structural/morphologic and metabolic changes,cell cycle regulation,etc.Erythropoiesis is synergistically regulated by extracellular signaling,intracellular transcriptional regulation,and epigenetic changes in chromatin,etc.Abnormalities in the regulation of erythropoiesis often lead to anemia,leukemia,and other diseases.Therefore,it is of great significance to study the regulation mechanisms of erythropoiesis in order to understand the underlying mechanisms of cell differentiation and disorders related to erythropoiesis.Transcription factors like GATA binding protein 1?GATA-1?and Erythroid Krüppel-like factor?EKLF/KLF1?play a key role in the regulation of erythropoiesis.Many proteins are involved in the erythropoiesis regulation by influencing their post-translational modifications,protein interactions,DNA binding,and transcriptional activities.G protein pathway suppressor 2?GPS2?has been extensively investigated during the inflammation,metabolism,and immunity process.But there's no research that has ever implicated that GPS2 might be involved in erythropoiesis.In this study,we investigated the role of GPS2 by using GPS2 knockout mouse model and human primary CD34⁺cells model and found that GPS2 is a new erythropoiesis regulatory factor playing an important role in erythropoiesis by stabilizing EKLF.The main results are listed below:1.The embryos of Gps2^-/-mouse at E12.5 showed typical hematopoietic development disorder phenotypes,such as pale embryo and small liver.The total number of E12.5 fetal liver cells and the Ter119⁺cell number in E12.5 fetal liver were significantly reduced.Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2^-/-embryos.2.GPS2 expression in human umbilical cord blood CD34⁺cells was significantly up-regulated by erythroid induction.Knockdown of GPS2 in CD34⁺cells resulted in impaired erythropoiesis,as assessed by decreased proportion of CD71⁺GPA⁺cells,benzidine positive cells,erythroid gene expression,and the number and proportion of burst-forming unit-erythroid?BFU-E?.Down-regulation of GPS2 also suppressed erythroid differentiation of human CD34+cells in vivo when xenotransplanted in NOD/SCID/IL2R?null?NSG?mice3.GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF,thereby increases EKLF stability and transcriptional activity.However,GPS2does not affect the ubiquitination of EKLF.GPS2 promotes erythroid differentiation in an EKLF-dependent but nuclear receptor corepressor 1?NCOR1?-independent manner in mouse erythroleukaemia?MEL?and human CD34⁺cells.GPS2 lacking the NCOR1binging region?amino acid?aa?1-110?still has promotive effects on erythropoiesis,while deletion of EKLF binding region?aa 111-150?makes GPS2 unable to promote erythropoiesis.Importantly,re-expression of EKLF completely rescues the inhibited erythroid differentiation in GPS2 knockdown cells.Notably,we identify the aa 191-230region in EKLF protein,responsible for GPS2 binding,is highly conserved in mammals and essential for EKLF protein stability,which may account for certain hematologic disorders caused by EKLF mutation.EKLF is a key transcription factor of erythropoiesis,and its knockout or mutation can lead to a severe disorder of erythropoiesis.The function of EKLF is regulated in many ways,but the regulation of its protein stability,especially the relationship with erythropoiesis,has not been reported yet.In recent years,the relationship between EKLF and human erythropoiesis disorder has received extensive attention.A variety of EKLF mutants have been found in different diseases,and some of them significantly affect the protein level of EKLF.Therefore,our study not only found a new erythropoiesis regulator,but also provided new ideas for revealing the pathogenesis of blood diseases related to EKLF.