Etude de la différenciation érythroïde des syndromes myélodysplasiques de faible risque (rôle des protéines GATA-1 et Hsp70)

PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

A short Gfi-1B isoform controls erythroid differentiation by recruiting the LSD1-corest complex through the dimethylation of its SNAG domain

Gfi-1B is a transcriptional repressor essential for the regulation of erythropoiesis and megakaryopoiesis. Here we identify Gfi-1B p32, a Gfi-1B isoform, as essential for erythroid differentiation. Gfi-1B p32 is generated by alternative splicing and lacks the two first zinc finger domains of the protein. Selective knock down of Gfi-1B p32 compromises erythroid differentiation, whereas its ectopic expression induces erythropoiesis in the absence of erythropoietin. Gfi-1B p32 isoform binds to Gfi-1B target gene promoters and associates with the LSD1-CoREST repressor complex more efficiently than the major Gfi-1B p37 isoform. Furthermore, we show that Gfi-1B includes a KSKK motif in its SNAG domain, which recruits the repressor complex only when dimethylated on lysine 8. Mutation of lysine 8 prevents Gfi-1B p32-induced erythroid development. Our results thus highlight a key role for the alternatively spliced Gfi-1B p32 isoform in erythroid development

Imatinib induces the differentiation of human normal erythroblasts and decreases cell surface expression of c-Kit.

<p>Imatinib (2 µM) was added for 4 days to a culture of CD34+ cells-deriving erythroblasts. <b>A. Effect of imatinib on erythroblast differentiation.</b> Left panel: Flow cytometry for CD71 and GPA markers. Right panel: May Grunewald Giemsa staining at Day 14 (d14).<b> B. Effect of imatinib on c-Kit expression</b> Left panel: Flow cytometry for CD117 and GPA. Right panel: Immunoblot for c-Kit. <b>C. Expression of functional c-Kit at cell surface.</b> UT-7/Epo cells were incubated with 2 µM imatinib, or vehicle at 37°C for indicated times, and labeled with 200 ng/ml [¹²⁵I]SCF or 1 UI/ml [¹²⁵I]Epo for 90 min at 4°C. Cell surface-associated radioactivity was counted. Results are the mean of at least three independent experiments. <b>D. UT-7/Epo cell proliferation in response to SCF.</b> UT-7/Epo cells were incubated with 1 UI/ml Epo or 50 ng/ml SCF, in the presence or absence (vehicle) of 2 µM imatinib (IM). At the indicated times, cells were counted in triplicates.</p

c-Kit internalization occurs independently of c-Cbl.

<p><b>A. Cell surface expression of c-Kit after SCF binding.</b> UT-7/Epo either non transfected (UT-7), or transfected with WT c-Cbl (WT) or 70Z c-Cbl mutant (70Z) were cultured with 1 UI/ml Epo (white bars), starved and then stimulated for 5 min with 250 ng/ml SCF (hatched bars). C-Kit cell surface expression was determined by flow cytometry. Results were expressed as RFI of CD117 normalized to cells in Epo alone and are the mean of three independent experiments. <b>B. Cell surface expression of c-Kit after imatinib treatment.</b> Parental, WT c-Cbl and 70Z c-Cbl-UT-7/Epo cells cultured with 1 UI/ml Epo (white bars) were treated for 24 h with 2 µM imatinib (grey bars). C-Kit cell surface expression was determined by flow cytometry and the results were expressed as RFI of CD117 normalized to cells in Epo alone and are the mean of three independent experiments. <b>C. Immunoblot analysis of c-Kit after imatinib treatment.</b> Parental, <i>wt</i>-c-Cbl and 70Z c-Cbl-UT-7/Epo cells were incubated with 2 U/ml Epo in the presence or absence of 2 µM IM for 24 h. Immunoblots using anti-c-Kit, c-Cbl and HA antibodies. Actin is used as loading control. Results are representative of three independent experiments.</p

Imatinib or masitinib decreases cell surface expression of mature c-Kit in UT-7/Epo cell line.

<p><b>A. Dose-dependent inhibition of c-Kit expression by TKI.</b> Cell surface expression of c-Kit. UT-7/Epo cells were incubated with SCF 50 ng/ml for 10 min (hatched bars) or indicated concentrations of imatinib (IM) (grey bars) or masitinib (MA) (black bars) for 4 h. <b>B. Kinetic of c-Kit down-regulation by TKI.</b> UT-7/Epo cells were incubated with 2 µM IM (grey bars) or MA (black bars) or 50 ng/ml SCF (hatched bars) for indicated times. <b>C-Kit cell surface expression was determined by flow cytometry.</b> Results were expressed as ratios of median fluorescence intensity (RFI), and the values were normalized to untreated cells. Results are the mean of at least three independent experiments.</p

Imatinib or masitinib induces c-Kit internalization and its degradation by lysosome pathway.

<p><b>A.</b> For analysis of internalization, UT-7/Epo cells were pre-incubated with 50 µM of methyl-β-cyclodextrine (MβCD) or vehicle (V) for 30 min then treated during 4 hours with imatinib (grey bars) or masitinib (black bars) or Epo alone (white bars). <b>B.</b> For the analysis of proteasomal degradation, UT-7/Epo cells were incubated with 1 UI/ml of Epo or 50 ng/ml of SCF (hatched bars), and 50 µM of LLnL (L) or vehicle (V) for 20 min at 37°C before treatment with 2 µM IM (grey bars) or MA (black bars) or Epo alone (white bars) for 4 h. <b>C.</b> For the analysis of lysosomal degradation, UT-7/Epo cells were incubated with 1 UI/ml Epo or 50 ng/ml SCF, and 100 µM methylamine (M) or vehicle (V) for 20 min before treatment with 2 µM IM (grey bars) or MA (black bars) or Epo alone (white bars) for 4 h. Expression of c-Kit by flow cytometry (cell surface) and by immunoblot. Actin was used as loading control. Data are representative of three independent experiments and the quantification of one experiment is shown.</p

Imatinib prevents c-Kit receptor re-expression after SCF-induced down-regulation.

<p><b>A. Down-regulation of c-Kit by imatinib is reversible.</b> UT-7/Epo cells were incubated with 50 ng/ml SCF (dotted line) or with 2 µM imatinib (filled line) for 2 h. After washing in PBS, cells were cultured with 1 UI/ml Epo for 24 h. CD117 expression was measured at the indicated times by flow cytometry and results were expressed as RFI. <b>B. Imatinib prevents the re-expression of functional c-Kit after SCF binding.</b> UT-7/Epo cells were pre-incubated with 50 ng/ml SCF. After washing in PBS, SCF-treated cells were re-seeded with 1 UI/ml Epo in the presence or absence of 2 µM IM for 24 h. Cells cultured with 1 UI/ml Epo in the presence or absence of IM were used as controls. At indicated times, cells were labeled with 200 ng/ml [¹²⁵I]SCF for 90 min at 4°C and the radioactivity was determined. Results were expressed as percentages of [¹²⁵I]SCF binding to Epo alone at time 0 and are the mean (± SD) of three independent experiments.</p