Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex

Abstract

Background: The human hemoglobin switch (HbF-->HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor.

Design and methods: We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34(+) cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production.

Results: In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80-90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects.

Conclusions: Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221-222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.

Figure 1.

Kit/KL expression in CB CD34⁺ cells in perinatal life. (Top). Kit expression levels in 61 CB and 20 control APB samples, as related to gestational age (A) and reticulocyte HbF content (B). The best fitting curves for CB values are presented. The curves are extrapolated to the mean adult value. The inverse correlation between kit level and gestational age and the direct one between kit level and HbF content are highly significant (P<0.001 and P<0.001, respectively). (Bottom). Plasma KL levels in CD34⁺ cells from 64 CB samples and 20 control APB samples, as related to gestational age (C) and reticulocyte HbF content (D). The best fitting curves for CB values are presented. The left panel curve is extrapolated to the mean adult value. The inverse correlation between KL level and gestational age, and the direct one between KL level and reticulocyte HbF content are highly significant (P<0.001 and P<0.01, respectively).

Figure 2.

Reactivation of HbF synthesis in CB erythroid culture. Growth curve (A) and percentage of mature (polychromatophilic + orthochromatic) erythroblasts (B) of minibulk HPC erythroid cultures supplemented or not with KL (1, 10 and 100 ng/mL). C indicates control cultures. Mean ± SEM values of 8 separate experiments. (C) Percentage of HbF content in whole blood RBCs, reticulocytes and mature erythroblasts from minibulk HPC erythroid cultures supplemented or not with KL (1, 10 and 100 ng/mL). Results from 8 pre-term and full-term CB samples are shown. Each experiment is represented by a different symbol; mean values are indicated. ***P<0.001 and 0.01 when compared to RBC and reticulocyte group respectively. **P<0.01 when compared to RBC group but not to reticulocyte group. N.S. (not significant) when compared to RBC and reticulocyte groups.

Figure 3.

Effect of imatinib (IM) on HbF reactivation in CB erythroid culture. (A) Growth curve of minibulk HPC erythroid cultures supplemented or not with KL (100 ng/mL)±Gleevec (2x10⁻⁶M). Mean ± SEM values from 5 separate experiments. (B) Percentage of mature (polychromatophilic + orthochromatic) erythroblasts in the erythroid cultures. (C) Percentage of HbF content in mature erythroblasts. (D) Left panel. Percentage of γ-globin chains in single sibling BFU-E colonies grown in unilineage erythroid cultures supplemented with KL (1, 10 and 100 ng/mL) ± Im (2x10 ⁻⁶M). Results from 5 experiments, each including 4 sibling colonies are presented (each experiment represented by symbol; mean values are indicated). Right panel. Cell number/colony values from 5 different clones, each including 4 sibling BFU-E colonies. **P<0.01, *P<0.05, N.S. (not significant) when compared to the indicated groups.

Figure 4.

Analysis of miR-221/222 expression in CD34⁺ HPCs. (A) miR-221/222 expression in pre-term CBs (white), mid-full-term CBs (gray) and APB (black). Fold increase of miR-221 and miR-222 levels as compared to pre-term CBs (set as 1) is indicated. Mean ± SEM (n=9 pre-term CB; n=12 mid-full-term CB; n=20 APB). ***P<0.001 when compared to pre-term CB group, **P<0.01 when compared to mid-full-term group. (B) Growth curve (left panel) and percentage of HbF (right panel) in HPC erythroid cultures untransfected (C) or transfected with a control miR (CmiR) or with miR-221 and miR-222 alone or in combination (miR-221+222). Cells were grown in absence (upper panels) or in the presence (lower panels) of KL. Mean ± S.E.M. (n=3). (Upper panels) P<0.05 when miR-221 and -222 growth curves are compared to C groups (left panel) and **P<0.01 when miR 221 and -222 HbF contents are compared to C groups (right panel). (Lower panels) P<0.01 when miR-221 and -222 growth curves are compared to C groups (left panel) and ***P<0.001 when miR 221 and -222 HbF contents are compared to C groups (right panel). (C) Growth curve (left panel) and percentage of HbF (right panel) in HPC erythroid cultures untransfected (C) or transfected with a control antagomiR (C antagomiR), antagomiR 221 or antagomiR 222. Cells grown in the presence of KL are included (KL). Mean ± SEM (n=3). **P<0.01 when compared to C groups (right panel). (D) Growth curve (left panel) and percentage of HbF content (right panel) in HPC erythroid cultures untransfected (C) or transfected with c-kit siRNA and grown in the presence of KL. Cells grown in the presence of KL alone or in combination with imatinib are included as positive and negative controls of HbF induction. Mean ± SEM (n=3). P<0.001 when KL+ siRkit growth curve is compared to KL curve (left panel) and ***P<0.001 when compared to KL HbF content (right panel).

Figure 5.

Effect of miR-221/222 overexpression/knockdown on kit levels. (A) Kit protein levels evaluated by Western blot in HPC erythroid cultures untransfected, C, or transfected with a control miR (C miR) or with miR-221 plus miR-222 (miR-221+222). Cells grown in the presence of KL are included (KL). Mean ± SEM (n=3). (B) miR-221/222 expression and (C) kit protein levels in HPC erythroid cultures untransfected (C) or transfected with a control antagomiR (C antagomiR) or with antagomiR 221 and antagomiR 222 alone or in combination (antagomiR 221+222). Cells grown in the presence of KL are included (KL). Mean ± SEM (n=3). ***P<0.001 and **P<0.01 when compared to C groups. (D) A representative Western blot analysis of the kit protein evaluated in erythroid HPC cultures 96 h after transfection with a control miR (C miR), control antagomiR (C antagomiR), miR-221 plus miR-222 (miR-221+222) or antagomiR 221 plus antagomiR 222 (antagomiR 221+222), respectively. β-actin controls are also included.