Abstract
TSPO2 (translocator protein 2) is a transmembrane protein specifically expressed in late erythroblasts and has been postulated to mediate intracellular redistribution of cholesterol. We identified TSPO2 as the causative gene for the HK (high-K+) trait with immature red cell phenotypes in dogs and investigated the effects of the TSPO2 defects on erythropoiesis in HK dogs with the TSPO2 mutation and Tspo2 knockout (Tspo2−/−) mouse models. Bone marrow–derived erythroblasts from HK dogs showed increased binucleated and apoptotic cells at various stages of maturation and shed large nuclei with incomplete condensation when cultured in the presence of erythropoietin, indicating impaired maturation and cytokinesis. The canine TSPO2 induces cholesterol accumulation in the endoplasmic reticulum and could thereby regulate cholesterol availability by changing intracellular cholesterol distribution in erythroblasts. Tspo2−/− mice consistently showed impaired cytokinesis with increased binucleated erythroblasts, resulting in compensated anemia, and their red cell membranes had increased Na,K-ATPase, resembling the HK phenotype in dogs. Tspo2-deficient mouse embryonic stem cell–derived erythroid progenitor (MEDEP) cells exhibited similar morphological defects associated with a cell-cycle arrest at the G2/M phase, resulting in decreased cell proliferation and had a depletion in intracellular unesterified and esterified cholesterol. When the terminal maturation was induced, Tspo2−/− MEDEP cells showed delays in hemoglobinization; maturation-associated phenotypic changes in CD44, CD71, and TER119 expression; and cell-cycle progression. Taken together, these findings imply that TSPO2 is essential for coordination of maturation and proliferation of erythroblasts during normal erythropoiesis.
Highlights
translocator protein 2 (TSPO2) is a transmembrane protein expressed in late erythroblasts and has been postulated to mediate intracellular redistribution of cholesterol
The findings from the present study revealed an important in vivo functional role for TSPO2 in both maturation and proliferation of late-stage erythroblasts
Considering the reduced cell proliferation and hemoglobinization in Tspo2Ϫ/Ϫ mouse embryonic stem cell– derived erythroid progenitor (MEDEP) cells and anemic phenotypes in Tspo2Ϫ/Ϫ mice, the TSPO2 defect may reduce the efficiency of red blood cell (RBC) production by ϳ50%
Summary
Genome-wide linkage analysis was conducted on seven HK and 17 LK dogs, including 15 dogs from two independent families of Japanese mongrel dogs (Fig. 1A). To examine whether C40Y and VFT mutations impaired the function of TSPO2, we analyzed the intracellular cholesterol distribution in K562 cells stably expressing the WT or the mutant cTSPO2 These cell lines integrated the transfected cDNAs at nearly equivalent levels (Fig. 3A). No apparent difference was observed among these cell lines in the uptake of transferrin (Fig. 3B), which shares the clathrin-mediated endocytosis pathway with cholesterol for incorporation into the cell [27] These data suggest that cTSPO2 induces specific alterations in intracellular distribution of cholesterol and that the cTSPO2 mutants failed to induce such redistribution. These data suggest that the TSPO2 function involves, in part, an accumulation of cholesterol in the ER and that the C40Y and VFT mutations are detrimental to this function
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