Mechanism Of CREG1 Regulation Of Megakaryocyte Differentiation And Thrombopoiesis

BackgroundPlatelets are small,disk-shaped cells with a diameter of 2-5μm.A large number of organelles and secretory granules are found in the cytoplasm of platelets.Platelet is an important part of circulating blood in the body.Its function is mainly reflected in maintaining the dynamic balance of bleeding and hemostasis,and participating in the pathophysiological processes such as tissue repair,inflammatory response and tumor growth.Megakaryocytes are derived from hematopoietic stem cells and are the precursor cells of platelets.They are also the only polyploid cells in human bone marrow,accounting for 0.05%of the total bone marrow nucleated cells.Megakaryocytes are divided into:granular megakaryocytes,platelet-producing megakaryocytes and naked nuclear megakaryocytes.After undergoing proliferation,differentiation and polyploidization,one megakaryocyte can produce almost 1000～6000 platelets.Aplastic anemia,bone marrow space-occupying diseases,viral infections,drugs,organic chemical reagents,and radiation can lead to the decrease of the generation and differentiation of megakaryocytes in bone marrow,and ultimately lead to thrombocytopenia.Recent studies have shown that dysfunctions of megakaryocyte differentiation and preplatelet formation can cause severe platelet-related diseases.However,the mechanism that regulates megakaryocytes differentiation and platelet formation still need to be further explored.CREG1（cellular repressor of E1A-stimulated genes 1）,is a small molecular weight secreted glycoprotein widely expressed in mature tissue cells and has the ability to maintain tissue and cell maturity.CREG1 protein is composed of 220 amino acids,contains 3 M6P（mannose 6 phosphate）glycosylation sites,and can interact with M6P receptors.Studies have found that CREG1 was highly expressed in mouse heart and blood vessel tissues.In addition,Gill et al.found that CREG1 was widely expressed in mature tissues,but very low in undifferentiated tissues,such as teratoma cells.Veal et al.introduced CREG1 protein into teratoma cells cultured in vitro and found that CREG1protein could inhibit cell proliferation and promote the spontaneous differentiation of teratomas into nerve cells.The results suggest that CREG1 may be a homeostatic regulator that induces cell differentiation.Further studies have found that CREG1 could maintain vascular homeostasis,promote endothelial recovery,and inhibit vascular fibrosis caused by hypertension.The study also found that Creg1 gene-specific knockout mice in hepatocyte caused obesity,glucose and lipid metabolic disorders due to the lack of Creg1.In addition,the exogenous recombinant CREG1 protein can improve the cardiac function of mice after myocardial ischemia-reperfusion injury by regulating the autophagy pathway.It can be seen that CREG1 could play an important role in the regulation of cardiovascular homeostasis and provide new clues and ideas for preventing and treating cardiovascular remodeling.Recently,we found abnormal differentiation and dysfunction of monocyte macrophages in mouse models with specific deletion of Creg1 gene;in addition,CREG1was highly expressed in platelets and megakaryocytes of human and mouse,and CREG1expression was decreased in platelets in thrombocytopenia patients.However,whether CREG1 can promote megakaryocyte differentiation and platelet formation remains unclear.The purpose of our research is to clarify the mechanism of CREG1 in the regulation of megakaryocyte differentiation and thrombopoiesis.CREG1 expression in platelets was decreased in patients with thrombocytopenia;further,using cytosine arabinoside（Ara-c）to induce thrombocytopenia in mice,it was found that the decrease of CREG1 expression in megakaryocytes and platelets was associated with thrombocytopenia.Using megakaryocyte/platelet-specific Creg1 knockout mice(Creg1^-/-)and Creg1 transgenic mice（tg-Creg1）as research models,starting from megakaryocyte differentiation and the generation of platelets,it was found that the number of platelets in Creg1^-/-mice was reduced,and the differentiation of megakaryocytes was inhibited.In a cell model where phorbol 12-myristate 13-acetate（PMA）stimulated the differentiation of Dami cells,CREG1 regulated extracellular signal-regulated kinase 1/2（ERK1/2）signaling pathway to promote megakaryocytes differentiation and polyploidization.In order to further clarify the mechanism of CREG1 regulation of megakaryocyte differentiation,we determined the core promoter of h CREG1 gene,and applied the dual-lusiferase report assay（Lusiferase）to discover that C/EBPβ（CCAAT enhancer binding protein beta）combined with the core promoter region of h CREG1,significantly up-regulated the activity of the core promoter of h CREG1,and promoted the differentiation of megakaryocyte.This study reveals the mechanism of CREG1 regulation of megakaryocyte differentiation and thrombopoiesis,and provides a theoretical basis for the prevention and treatment of thrombocytopenia.Methods（1）Establishment of thrombocytopenia model:Intraperitoneal injection of Ara-c 200 mg/kg/day for 2 days and 50 mg/kg/day for 3 days,and the number of blood cells was measured on the 5th day and the 10th day,respectively.On the 10th day,the number of platelets still decreased,while the white blood cells and red blood cells returned to normal.The thrombocytopenia model established by this dose of Ara-c was relatively stable.（2）Construction of Creg1^-/-and tg-Creg1 mice:Use Cas9/RNA system gene targeting technology to construct megakaryocyte/platelet Creg1^-/-mouse model.The tg-Creg1 mouse model was prepared by pronuclear injection.（3）Detection of platelet lifespan in mice:Dy Light 488 fluorescently-labeled rabbit-anti-GPIX（CD42a）antibody was injected into mice by tail vein injection,and blood was taken before and after injection at different time points.Cytometry was used to detect the lifespan of platelets in different groups of mice and observed the platelet clearance rate.（4）Detection of platelet production in mice:The rabbit-anti-GPIbα（CD42b）antibody was injected into mice by tail vein injection.The number of platelets were measured before and after the injection,and the platelet production curve was observed.（5）TPO intervention:TPO was injected into mice by tail vein injection,and the number of platelets were measured before and on the 4th day after injection.（6）Induction and isolation of bone marrow megakaryocytes:After extracting the cells in the bone marrow,resuspend the cells in DMEM medium containing 10%FBS, added recombinant human stem cell factor（SCF）and recombinant TPO,and applied BSA gradient centrifugation method to obtain megakaryocytes.（7）Induction and isolation of fetal liver megakaryocytes:We selected pregnant mice with a gestational age of 13.5 to 15 days,took out the fetal liver,grinded,filtered and other operations,then the cells were cultured in a 5%CO₂,37℃incubator for 4～5 days,added the same dose of TPO once a day.Megakaryocytes can be obtained by BSA gradient centrifugation.（8）Pre-platelet production test:We used fibrinogen（Fg）to coat sterile glass slides, planted the megakaryocytes on the glass slides,cultured in an incubator at 37°C with 5% CO₂.We observed the morphological changes of megakaryocytes under a microscope. Immunofluorescence staining was used to identify the formation of pre-platelets.（9）Lusiferase activity detection:We constructed h CREG1 core promoter fragments （including mutant fragments）expression vectors and transcription factor plasmids,and used lusiferase to detect the regulation of transcription factors on h CREG1 core promoter activity.Results（1）Loss of CREG1 led to a decrease in the number of platelets,namely the occurrence of thrombocytopenia.（2）The lack of CREG1 led to platelet production decreased,rather than increased platelet clearance in the peripheral blood.（3）The lack of CREG1 caused extramedullary hematopoiesis and splenomegaly.（4）Loss of CREG1 inhibited megakaryocyte differentiation and pro-platelet production.（5）CREG1 regulated the ERK1/2 signaling pathway to promote megakaryocyte differentiation and polyploidization.（6）The transcription factor C/EBPβregulated the activity of the core promoter of h CREG1.ConclusionIn summary,CREG1 may be involved in megakaryocyte differentiation and platelet production as a new regulatory factor.As a transcription factor,C/EBPβbinds to the core promoter region of h CREG1 and further activates the ERK1/2 signaling pathway by affecting the activity of the core promoter of h CREG1 to promote megakaryocyte differentiation and platelet production.As a key target,CREG1 provides a theoretical basis and new ideas for the prevention and treatment of thrombocytopenia.