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Tmem30a in erythropoiesis and EPOR signaling
into mature blood cells in the fetal liver.2 During embryo- genesis, primitive erythroid cells (EryP) first arise from mesodermal progenitors and are detected within 'blood islands' at around E7.5. The maturation of erythroid pre- cursors occurs in the blood circulatory system, where the nucleuses are condensed and embryonic hemoglobin is accumulated.3 Definitive erythroid cells (EryD) rapidly outnumber the EryP in the growing fetal liver,4,5 which are identified as β-globin switching and smaller enucleated erythroid cells.6 The fetal liver is the key organ for definitive erythropoiesis during mid gestation. Definitive erythroid cells can be dis- tinguished into five different sub-populations from R1 to R5 by double staining with the surface markers CD71 and Ter119.7 Erythropoiesis comprises distinct differentiation stages including burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte and erythro- cyte. From the CFU-E stage onwards, the cell starts to express erythropoietin (EPO) receptor (EPOR). CFU-E and proerythroblat require EPO for survival.8
Erythroid differentiation occurs at the erythroblastic islands and is regulated by various cytokines and chemokines. EPO and stem cell factor (SCF) play essential roles in erythroid progenitor proliferation and differentia- tion. EPO is mainly synthesized in liver during embryo genesis and produced in the kidney in adult mammals. EPO/EPOR-mediated signaling transduction is crucial for primitive and definitive erythropoiesis both in the fetal liver (FL) and in the bone marrow.9 EPO has two receptors: one is a homodimer of two EPO receptors (EPOR), anoth- er is a heterodimer consisting of EPOR and CD131.10 The homodimeric EPO receptor exists in an unliganded state with the pre-bound tyrosine kinase JAK2.11 Upon binding EPO, EPOR undergoes a conformational change that actives JAK2 which in turn phosphorylates tyrosine residues in the cytoplasmic tail of the EPOR.12 This bind- ing results in activation of STAT5, which leads to the acti- vation of BCL-XL by direct STAT5 binding to the BCL-X promoter.13 BCL-XL is a potent inhibitor of programmed cell death and inhibits activation of caspases in cells through direct interaction between caspases and BCL- XL.14,15 The activation of the JAK2-STAT5 pathway through EPO/EPOR signaling is critical for sustaining the viability of erythroid cells in the fetal liver.16
Lipid rafts are small microdomains (10-200 nm) enriched in cholesterol and sphingolipids that can form larger platforms by protein-protein and protein-lipid inter- actions. The inner leaflet phosphatidylserine is essential for the coupling of actin with lipid-anchored proteins. The actin cytoskeleton clustering determines and immobilizes long saturated acyl chains phospholipids in the inner leaflet.17 This immobilization engages in glycosylphos- phatidylinositol (GPI)-anchored proteins in the outer monolayer interacted by cholesterol, which form the local raft domains. The most important role of lipid rafts is to separate and regulate specific membrane components with other components, thereby increasing the concentra- tion of signaling molecules.
In eukaryotic cells, phospholipids are distributed asym- metrically between the inner and the outer layers of the plasma membrane.18 Phosphatidylserine (PS) and phos- phatidylethanolamine (PE) are mainly located in the inner monolayer while phosphatidylcholine (PC) is essentially
present at the outer monolayer.19,20 Lipids distributions are preserved by many of phospholipid transporters which can be separated into three groups including scramblases, flippases and floppases.21 One of the most important trans- porters are the members of the Type-IV P-type ATPases (P4-ATPases) family which possess flippase activity that transports lipids from the outer to the inner leaflet to maintain phospholipid asymmetry. Tmem30a (also named CDC50A), the β-subunit of P4-ATPases, is essential for the formation of functional transporter complexes that act as flippase.22 Maintenance of cell membrane asymmetry by flippase is critical as the loss of this asymmetry usually causes pathological phenotypes.23
To investigate the function of Tmem30a in embryonic hematopoiesis, we generated hematopoietic-specific Tmem30a deficient mice with conditional Tmem30a alleles and Cre recombinase expression controlled by the VAV promoter.24 Tmem30a deficient mice (cKO) died in utero by E16.5 with severe anemia. Interestingly, Tmem30a is not essential for the maintenance of HSC homeostasis, but is essential for the definitive erythropoiesis. Moreover, Tmem30a deficiency impaired flippase activity, lipid rafts formation, and activation of EPOR/JAK2/STAT5/BCL-XL pathway. Our findings demonstrate the critical role of Tmem30a in erythropoiesis and uncover previously unknown mechanisms by which EPOR signal transduc- tion pathway is initiated.
Methods
Mice
All mouse protocols were approved by the Institutional Animal Care and Use Committee of Jinan University, China. Tmem30aWT/flox mice were kindly provided by Prof. Xianjun Zhu and were back-crossed onto a C57/BL6 background. Exon 3 of the Tmem30a gene is flanked by loxP sites. The Vav-Cre line we used was B6.Cg-Commd10Tg(Vav1-icre)A2Kio to generate hematopoietic deletion, as described previously.25
Flow cytometry
Cells were stained with APC-conjugated rat anti–mouse TER- 119 (clone: Ter119, Biolegend) and PE conjugated rat anti-mouse CD71 (clone: RI7217, Biolegend) on ice for 30 minutes (min) in the dark. The cells were washed twice, followed by staining with fix- able viability DAPI (0.25 μg/106 cells) and analyzed within 1 hour (h) of staining. For apoptosis, cells were additionally stained for 15 min in the dark with 10 μL of Annexin V-FITC in 100 μL 1xbind- ing buffer.26 Cells were washed and cell pellets were re-suspended in 500 μL 1xbinding buffer containing 5 μL of 7AAD and immedi- ately analyzed by a BD FACS Fortessa machine. Apoptosis was also assessed using TUNEL assay by flow cytometry using APO- BrdU TUNEL assay kit (Invitrogen, A23210).
Western blot analysis
Fetal liver cell samples were separated and then transferred to the polyvinylidene fluoride membrane. Primary antibodies to STAT5 (#9363, CST), Phospho-STAT5 (Tyr694) (#9351, CST), BCL-XL (54H6) (#2764, CST), Flotillin-2 (B-6) (SC-28320, Santa Cruz), TMEM30A (AV47410, Sigma-Aldrich), EPO-R (SAB4500780, Sigma-Aldrich) and β-Actin (A5316, Sigma-Aldrich) were used. The membrane was incubated with horseradish per- oxidase enzyme conjugated secondary antibody for 1 h at RT. Clarity western ECL substrate solutions were dropped onto the membrane.
haematologica | 2019; 104(10)
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