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XPO1 is a target to treat β-thalassemia
Introduction
Erythropoiesis is a multistep process leading to red cell production from hematopoietic stem cells. We have demon- strated that entry into the terminal maturation stage of ery- throblasts, marked by important morphological modifica- tions, requires a transient activation of caspase-3.1 However, in this process, some caspase-3 targets including GATA1, the transcription factor involved in the expression of erythroid genes like erythropoietin (EPO) receptor, Glycophorin A or globin chains, remains uncleaved. The chaperone Heat Shock Protein 70 (HSP70), which is constitutively expressed during erythroid differentiation, accumulates in the nucleus and protects GATA-1 transcription factor from caspase-3 cleavage, allowing continued terminal maturation.2 We have demonstrated that this nuclear accumulation of HSP70 did not occur in human β-TM erythroblasts, resulting in GATA- 1 cleavage and thus erythroblast maturation arrest and apoptosis. Indeed, in β-TM, HSP70 is sequestrated in the cytoplasm by the excess of free a-globin chains preventing its nuclear localization.3 Likewise, terminal maturation in human β-TM could be restored by transduction of a nuclear-targeted HSP70 mutant. Thus, any regulation enabling an increase in HSP70 nuclear concentration would be an interesting therapeutic strategy to correct ineffective erythropoiesis of β-TM. However, mechanisms involved in HSP70 trafficking during human erythroid differentiation are still unknown. Thus, we focused our work on elucidat- ing those mechanisms. We have previously shown by both confocal microscopy and immunoblot analyses that, in ery- throblasts derived from CD36+ cells, EPO starvation induced a dramatic decrease in both nuclear localization of HSP70 and as a consequence GATA-1 expression. Addition of the nuclear export inhibitor Leptomycin B (LMB)4,5 prevented this phenomenon.2 These previous observations strongly suggested that HSP70 localization could be regulated mainly by its export. Exportins are proteins of the β-karyopherin group that mediate export from the nucleus to the cyto- plasm of cellular proteins (cargos) or mRNAs, using the RanGTP/GDP gradient. There are seven known exportins expressed in human cells (XPO1 to XPO7) and all show pref- erential cargo specificity.6 It has been recently reported that the erythroid-specific isoform of exportin-7 (XPO7) was the most abundant exportin expressed at the mRNA level in very late erythropoiesis, and was involved in chromatin con- densation and enucleation in murine erythroid differentia- tion.7 However, since XPO7 is expressed and plays a role at the later stage of erythroid differentiation, it might not be considered as a good candidate to be an exporter of HSP70.
In order to decipher which exportin(s) was/were involved in HSP70 nuclear export in erythroid progenitor, we have analyzed the expression profile of the seven different human exportins from transcriptomic and proteomic data from erythroid cells at different differentiation stages of mat- uration, and showed functionally that XPO1 is involved in HSP70 export and could be considered as a good target to ameliorate ineffective erythropoiesis of β-TM.
Methods
Erythroid liquid culture and KPT-251 treatment
Erythroid cells were generated in vitro from peripheral blood cir- culating CD34+ cells from adult patients with β0-thalassemia major (β-TM), which were collected before routine transfusion or
from cord blood from healthy donors. This study was performed according to the Declaration of Helsinki with the approval from the ethics committee of our institution [Comité de Protection des Personnes (CPP) “Ile de France II”]. All patients gave written informed consent. In the first step of culture (“cell expansion”), iso- lated CD34+ progenitors (Miltenyi CD34 Progenitor Cell Isolation Kit) were grown in the presence of 100 ng/mL IL-6, 10 ng/mL IL- 3, and 100 ng/mL SCF for 7 days. At day 7, erythroid progenitors were switched to the second phase of culture, which allows the differentiation and maturation of erythroblasts: cells were cultured in the presence of 10 ng/mL IL-3, 50 ng/mL SCF, and 2 U/mL EPO in IMDM (Gibco cell culture) supplemented with 15% BIT 9500 (Stem Cell Technologies), as previously described. Solid KPT-251 was reconstituted in DMSO. Cells were treated with KPT-251 at 100 or 1000 nM, or with DMSO alone as control condition.
Cell differentiation
Erythroid differentiation was assessed by various methods. First, morphological analysis after May-Grunwald-Giemsa (MGG) staining was used. Cells were examined under Zeiss microscope axioplan 2, Camera Qimaging QICAM FAST 1394. The number of immature (proerythroblasts + basophilic), poly- chromatophilic, and mature (orthochromatic erythroblasts + reticulocytes) erythroblasts was assessed in each experiment by counting approximately 200 cells in consecutive fields and expressed as a percentage of total cells. Additionally, differenti- ation was assessed by calculating a terminal maturation index (TMI) on MGG, defined by the number of orthochromatic ery- throblasts + reticulocytes*100/ number of polychromatophilic cells per slide. This allowed us to better characterize the matu- ration arrest at the polychromatophilic stage, which is known to be a hallmark of β-TM ineffective erythropoiesis,8 and its modulation.
Flow cytometry analysis was also performed at several differ- ent times during erythroid culture after double labeling with Band3 and 4-integrin, two optimal surface markers to differen- tiate highly mature erythroblasts.9 PE-conjugated anti-Band3 (PE-BRIC6 conjugate, Bristol Institute for Transfusion Sciences, UK) and APC-conjugated anti- 4-integrin (Miltenyi Biotec) anti- bodies were used for flow cytometry (FACS Gallios). This dou- ble labeling allowed us to assess terminal erythroblastic differ- entiation and purify cell populations by cell sorting (BD FACS Aria II SORP). High Band3 (high band3, low a4-integrin), and low Band3 (low band3, high 4-integrin) gates were defined, and cells from each gate were sorted and analyzed morpholog- ically after MGG staining.
Statistical analysis
Statistical analyses were performed with GraphPadPrism (Version 4.0 GraphPad software). Data are expressed as the mean±standard deviation or standard error of the mean. Paired t-test, or Anova Dunn’s, Dunnett’s, and Tukey’s multiple com- parison tests were used as appropriate. P<0.05 was considered statistically significant; lower P-values are indicated in the fig- ure legends. Further information is available in the Online Supplementary Appendix.
Results
XPO1 can interact with HSP70 nuclear export sequence
In our previous work,2 we demonstrated that HSP70 export mediated by EPO deprivation could be repressed by treatment with the nuclear export inhibitor LMB.
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