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, 284 (44), 30484-97

Translocator Protein 2 Is Involved in Cholesterol Redistribution During Erythropoiesis

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Translocator Protein 2 Is Involved in Cholesterol Redistribution During Erythropoiesis

Jinjiang Fan et al. J Biol Chem.

Abstract

Translocator protein (TSPO) is an 18-kDa cholesterol- and drug-binding protein conserved from bacteria to humans. While surveying for Tspo-like genes, we identified its paralogous gene, Tspo2, encoding an evolutionarily conserved family of proteins that arose by gene duplications before the divergence of avians and mammals. Comparative analysis of Tspo1 and Tspo2 functions suggested that Tspo2 has become subfunctionalized, typical of duplicated genes, characterized by the loss of diagnostic drug ligand-binding but retention of cholesterol-binding properties, hematopoietic tissue- and erythroid cell-specific distribution, and subcellular endoplasmic reticulum and nuclear membrane localization. Expression of Tspo2 in erythroblasts is strongly correlated with the down-regulation of the enzymes involved in cholesterol biosynthesis. Overexpression of TSPO2 in erythroid cells resulted in the redistribution of intracellular free cholesterol, an essential step in nucleus expulsion during erythrocyte maturation. Taken together, these data identify the TSPO2 family of proteins as mediators of cholesterol redistribution-dependent erythroblast maturation during mammalian erythropoiesis.

Figures

FIGURE 1.
FIGURE 1.
TSPO2 is a new subfamily of TSPO proteins. A, NJ trees of TSPO proteins from three domains of life organisms, rooted with TSPO proteins from bacteria. B, NJ trees of TSPO and TSPO2 proteins from vertebrates rooted with TSPO from fish. TSPO2 proteins from avians to mammals are in a separate group from the rest of the TSPO proteins with a strong bootstrap support (930/900/970 in 1000 replicates), whereas TSPO proteins are widely distributed within three domains of life. The numbers at the nodes to assess the robustness of the trees represent the NJ bootstrap value, the maximum likelihood bootstrap value, and the maximum parsimony bootstrap value, respectively. Only bootstrap values larger than 50% are shown, and the boldface letters indicate the main branches. The estimated genetic distance indicated as the number of substitutions per amino acid site is proportional to the horizontal length of each branch. GenBankTM accession numbers and other abbreviations used are listed in supplemental Table S1. C and D, comparison of the hydropathy plots of the predicted amino acid sequences of human and mouse TSPO/TSPO2. The hydropathicity indices were determined as described (53). The shaded bars below the plots indicate transmembrane domains predicted by the TMpred algorithm (24). Hydrophobic residues are positive.
FIGURE 2.
FIGURE 2.
Subcellular localization of mouse and human TSPO2 proteins in cultured mouse and human cells. GFP-tagged mTSPO2 and hTSPO2 were cotransfected with RFP-tagged mTSPO1 into NIH 3T3 (A–F), MA-10 (G–L), or HeLa (M–R) cells. The ER membrane was labeled with ER-Tracker Blue-White DPX. A, G, and M, GFP; B, H, and N, ER-Tracker Blue-White DPX; C, I, and O, GFP and ER-Tracker merged; D, J, and P, RFP; E, K, and Q, GFP and RFP merged; F, L, and R, GFP, RFP, and ER-Tracker merged images. Bars, 5 μm.
FIGURE 3.
FIGURE 3.
Characterization of [3H]PK 11195- and cholesterol-binding properties of recombinant TSPO proteins in yeast cells. A, expression of human and mTSPO2 and mTSPO1 proteins in S. cerevisiae INVSc1. Shown is Western blot analysis of total yeast proteins from strains transformed with plasmid pYES3/CT-mTSPO2, pYES3/CT-hTSPO2, and pYES3/CT-mTSPO1 and induced with 2% galactose under different time points: 0 h (uninduced control), 8 h, and 12 h. Anti-His tag antibody and anti-mTSPO1 were used to monitor the induced heterologous expression of the recombinant proteins. B, SDS resistance oligomerization of recombinant mTSPO2 in S. cerevisiae. The same amount (6 μg) of mTSPO2 recombinant protein was loaded on each well, transferred onto polyvinylidene difluoride membrane, and then incubated with a serially diluted antiserum (third bleed) from rabbit 4483 immunized with mTSPO2 peptide (IHQPSSRCEDERKLPWC), and different sample preparations were carried out to check the oligomerization of mTSPO2, which is one of the features of TSPO proteins and might be related to the formation of dityrosines as the covalent cross-linker between each monomer (27). C, system test of PK11195 binding of mTSPO1 protein using strains containing vector alone and uninduced mTSPO1 as controls. In the assay, 20 μg of each yeast lysate and different concentrations of PK11195 were used as indicated. D–H, [3H]PK11195-binding properties of vector alone (D), mTSPO2 (E), hTSPO2 (F), and mTSPO1 (G). H, Scatchard plot of [3H]PK11195 binding in mTSPO1 is shown where specific binding of [3H]PK11195 was calculated as total binding (in the absence of competitor) minus nonspecific binding (determined in the presence of 10 μm PK11195). Radioligand concentrations were in the range of 0.1–15 nm; each point represents the average of triplicate determinations at each concentration. D–H, 15 μg of total yeast lysate was used in the binding assay. I, comparison of conserved signature cholesterol binding motif (CRAC) between TSPO2 and TSPO1. J, cholesterol uptake of S. cerevisiae spheroplasts with induced overexpression of mTSPO2, hTSPO2, and mTSPO1 and different concentrations of cholesterol, as indicated. The specific cholesterol uptake is defined as recombinant proteins induced minus vector alone basal values. K, NBD-cholesterol uptake assay. S. cerevisiae strains were induced with 2% galactose to overexpress mTSPO2 in the presence of NBD-cholesterol and under aerobic conditions (>O2). Vector alone cells were used as control. L, NBD-cholesterol uptake assay. S. cerevisiae strains were induced with 2% galactose to overexpress mTSPO2 in the presence of NBD-cholesterol and under anaerobic conditions (<O2). Vector alone was used as the control. Differential interference contrast images of the cells with less cholesterol uptake are shown, respectively, and another two differential interference contrast images are shown in supplemental Fig. S7.
FIGURE 4.
FIGURE 4.
Cholesterol binding of mTSPO2. A, the schematic structure of NH2 terminus hexahistidine-tagged recombinant proteins, His-mTSPO2-WT and His-mTSPO2-CRAC. B, solubility of the recombinant mTSPO2 proteins. Western blot analysis was performed to check different fractions of cell lysates. M, protein standards; Total, total cell lysate; Sol, soluble fraction; IB, purified inclusion bodies. C, [3H]cholesterol binding. Column-based affinity chromatography was used to test the [3H]cholesterol binding properties. WT, His-mTSPO2-WT; CRAC, His-mTSPO2-CRAC. Results shown are means ± S.E. form three independent experiments.
FIGURE 5.
FIGURE 5.
Tspo2 expression in mice. A, Tspo2 expression in the embryonic mouse (e10.5, e12.5, and e15.5). a, d, and g, anatomical view of an embryonic mouse after staining with cresyl violet. b, e, and h, x-ray film autoradiography following hybridization with antisense riboprobe after a 5-day exposure. There is no labeling evident at this e10.5 stage, but a pattern of Tspo2 mRNA distribution is shown to be restricted to hepatic primordium at the e12.5 stage and in the hepatic primordium and bone, including the ribs and alveolar bone in the mandibles at stage 15.5, seen as bright labeling under dark field illumination at the e12.5 stage. c, f, and i, control sense (s) hybridization of an adjacent section comparable with b, e, and h, respectively. B, Tspo2 mRNA in the newborn (p1). a, anatomical view of the p1 mouse after staining with cresyl violet. b, x-ray film autoradiography following hybridization with antisense riboprobe showing a pattern of Tspo2 mRNA labeling seen as bright labeling under dark field illumination. Tspo2 mRNA occurs in the liver, bone, and spleen. c, control sense (s) hybridization of an adjacent section comparable with b. C, Tspo2 mRNA in the postnatal (p5 and p10). a and d, anatomical view of the postnatal (p5 and p10) after staining with cresyl violet. b and e, x-ray film autoradiography following hybridization with antisense riboprobe showing a pattern of Tspo2 mRNA labeling seen as bright labeling under dark field illumination. Tspo2 mRNA occurs in the liver, in vertebrae bone marrow, and in alveolar bone at stage p5; however, Tspo2 mRNA apparently can only be seen in the vertebrae bone marrow and alveolar bone at stage p10. In comparison with the p5 stage, a dramatic decrease in gene 1 labeling is observed in the liver. c and f, control sense (s) hybridization of an adjacent section comparable with b and e, respectively. D, Tspo2 mRNA in the adult mouse (p77). a, anatomical view of the adult mouse, after staining with cresyl violet. b, Tspo2 mRNA labeling mainly in the vertebrae bone marrow and bone tissue, no labeling in the liver. c, control sense (s) hybridization of an adjacent section comparable with b. Note the relatively high sense labeling in the vertebrae bone marrow, molars, and alveolar bone (asterisks) in p10 and adult. E, Tspo2 expression in the liver development. a–g, emulsion autoradiography showing Tspo2 expression in the liver seen as silver grains under light field illumination at high magnification (×540) of tissue stained with cresyl violet. Developmental stages are indicated. The arrows point to the groups of labeled cells. h, control (sense) hybridization shown in the liver primordium on day 15.5. F, Tspo2 expression in the bone marrow development. a–f, emulsion autoradiography showing Tspo2 mRNA labeling (arrows) in the bone marrow under light field illumination at medium magnification (×220 and ×130), tissue stained with cresyl violet. Developmental stages are indicated. The arrows point to the groups of labeled cells. h, control (sense) hybridization shown in the adult bone marrow (p77). G, Tspo2 expression in the newborn (p1) and adult (p77) mouse spleen. a, e, and i, emulsion autoradiography showing Tspo2 mRNA labeling (arrow) in the newborn and adult mouse spleen at medium (×61) and high (×110) magnification, showing the white pulplike labeling pattern (arrow). b, f, and j, the same region as in a, e, and i seen with cresyl violet staining under bright field illumination. c and g, control (sense) hybridization in the adjacent section comparable with a and e, respectively. d and h, the same region as in c and g seen with cresyl violet staining under bright field illumination. AB, alveolar bone in the mandible; B, bone; BM, bone marrow; Br, brain; C, capsule; Cp, capillary; FL, forelimb; H, heart; He, hepatocyte; HL, hind limb; HV, heart ventricle; Li, liver primordium; Me, mesencephalon; Mk, megakaryocyte; N, nose; R, ribs; RP, red pulp; SC, spinal cord; Spl, spleen; St, stomach; Te, telencephalon; Ve, vertebrae; WP, white pulp; as, antisense; s, sense.
FIGURE 6.
FIGURE 6.
Tspo2 expression in bone marrow and spleen cells purified from flow cytometry at differentiation stages of erythroblast maturation. A and B, fluorescence-activated cell sorting for progenitor cells. Freshly dissociated wild-type mouse bone marrow and spleen cells were labeled with a fluorescein isothiocyanate-conjugated monoclonal antibody to CD71 and a phycoerythrin-conjugated anti-Ter119 monoclonal antibody. Dead cells (staining positive with propidium iodide, shown in the PerPC-Cy5-A channel) and anucleated red cells (with low forward scatter (FSC)) were excluded from analysis. The upper panels illustrate a density plot of all viable bone marrow and spleen cells; the axes indicate relative fluorescence units for phycoerythrin (x axis) and fluorescein isothiocyanate (y axis). The lower panel shows that regions SP1–SP6 were selected as indicated. These are predominantly proerythroblasts in region SP6, basophilic erythroblasts in region SP5, late basophilic and chromatophilic erythroblasts in region SP4, and orthochromatophilic erythroblasts (possibly containing some enucleated reticulocytes) in region SP3. SSC, side scatter. PE, phycoerythrin; FITC, fluorescein isothiocyanate; PI, propidium iodide (shown in the PerPC-Cy5-A channel). C and D, RT-PCR of mTSPO2 gene expression in sorted cells from each of regions SP6 to SP3 were performed using the total RNAs prepared from each purified cell subset of bone marrow (C) and spleen (D). Mouse tubulin and HPRT gene expression and unseparated cells from bone marrow or spleen were used as controls for RNA content per sample and positive gene transcripts, respectively. The Tspo1 was also presented in comparison with Tspo2.
FIGURE 7.
FIGURE 7.
Overexpression of hTSPO2 in human erythroid leukemia cell line K562 and redistribution of intracellular cholesterol. A, confirmation of the stably overexpressed hTSPO2 in cell line K562 by RT-PCR. Lanes 1 and 3, overexpression of hTSPO in stable cell line K562-hTSPO2; lanes 2 and 4, vector alone in stable cell line K562-vec. Detection of the human TSPO1 gene was used as positive control and the no-reverse transcription PCRs were used as negative control. Scale bar, 10 μm (left) and 50 pixels (right). B, redistribution of NBD-cholesterol uptake. KG, overexpression of hTSPO in the stable cell line K562-hTSPO2; KV, vector alone in stable cell line K562-vec; K562, wild-type parental cell line without transfection. Arrow, accumulated endosomal lipid droplets. C, three-dimensional graph to highlight the redistribution of free cholesterol from the cells with overexpression of hTSPO2, vector alone, and wild type. D, redistribution of intracellular free cholesterol as measured by filipin staining after the cells were treated with or without an inhibitor of desmosterol Δ24-reductase (U18666A). Scale bar, 10 μm.
FIGURE 8.
FIGURE 8.
Cholesterol biosynthetic pathway in erythroblasts. Main enzymes related to cholesterol synthesis and putative TSPO2-nuclear complex are listed with the ID number in the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array, as follows: squalene epoxidase (SQLE) (209218_at); lanosterol synthase (LSS) (202245_at); 3β-hydroxysteroid-Δ24-reductase (DHCR24) (200862_at); 3β-hydroxysterol Δ-(14)-reductase (C14SR, TM7SF2) (DHCR14A) (210130_s_at); hydroxysteroid (17β) dehydrogenase 10 (HSD17B10) (202282_at); 3β-hydroxysteroid-Δ5-desaturase (lathosterol dehydrogenase) (SC5D) (211423_s_at); 3β-hydroxysteroid-Δ7-reductase (DHCR7) (201791_s_at); lamin B receptor (LBR) (201795_at); lamin B2 (LBR) (201795_at); translocator protein 2 (TSPO2) (215449_at); translocator protein 1 (TSPO1) (202096_s_at). A heat map view of TSPO2 mRNA is shown below the profiling plot, where the largest values are displayed as the deepest red (hot), the smallest values are displayed as the deepest blue (cool), and intermediate values are a lighter shade of either blue or red. The red line represents the gene indicated at the left side of each profiling plot, and the remaining colors indicate the top four genes with the closest similar patterns of gene expression during the erythroid differentiation. These were predicted using Class Neighbors in GenePattern 3.11. D1–D11, the days in the time course of in vitro erythroid differentiation of human adult-derived peripheral blood CD34+ cells (GEO accession number GSE4655) (43).

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