Reduced GATA1 levels are associated with ineffective erythropoiesis in sickle cell anemia

Abstract

Ineffective erythropoiesis (IE) is defined as the abnormal differentiation and excessive destruction of erythroblasts i n the bone marrow, accompanied by an expanded progenitor compartment and relative reduction in the production of reticulocytes. It is a defining feature of many types of anemia, including β-thalassemia. GATA1 is an essential transcription factor for erythroid differentiation, known to be implicated in hematological conditions presenting with IE, including β-thalassemia and congenital dyserythropoietic anemia. However, little is known about the role of GATA1 in the erythropoietic defects recently described in sickle cell anemia (SCA). In the present study, we performed a detailed characterization of the role of GATA1 and ineffective erythropoiesis in SCA using both in vitro and in vivo assay systems. We demonstrate a significant decrease in GATA1 protein levels during SCA erythropoiesis and a concomitant increase in oxidative stress. Furthermore, we found that an increase in the activity of the inflammatory caspase, caspase 1, was driving the decrease in GATA1 levels during SCA erythropoiesis and that, upon inhibition of caspase 1 activity, SCA erythropoiesis was rescued and GATA1 levels partially restored. Our study further elucidates the defect in erythropoiesis in SCA, and may therefore help in the development of novel approaches to normalize the bone marrow niche prior to stem cell transplantation, or facilitate the production of healthy stem cells for gene therapy.

Figure 1.

Reactive oxygen species levels during erythroid differentiation in healthy donor and sickle cell disease -SS genotype. (A) A histogram showing the levels of reactive oxygen species (ROS) in healthy donor (HD) (red) and sickle cell anemia (SS) (blue) cells at day 2 (D2), D4, D6, D8 of differentiation. (B) A line graph representing the mean fluorescence intensity (MFI) of ROS staining in HD and SS cells at D2, D4, D6, D8 of differentiation (N=4), (mean ± standard deviation). *P<0.05, Mann-Whitney test.

Figure 2.

GATA1 protein levels in differentiating healthy donor and sickle cell disease -SS genotype primary cells. (A) A histogram showing the analysis by flow cytometry of glycophorin A-postive (GPA⁺) cells, assaying for GATA1 protein on day 2 (D2) and D6 of differentiation of healthy donor (HD) (red) and sickle cell anemia (SS) (blue) cells. (B) Bar graph representing the mean fluorescence intensity (MFI) of GATA1 in the GPA⁺ population at D6 and D8 of HD (red) and SS (blue) cell differentiation (N=4) (mean ± standard deviation) [SD]. (C) Western blot images of GATA1 (upper panel) and actin (lower panel) proteins in nuclear extracts of HD and SS cells at D6 and D8 of differentiation. (D) Bar graph representing the relative quantification by western blotting of GATA1 protein levels in the nuclear extracts of HD and SS cells at D6 and D8. Protein loading per lane for quantification was adjusted relative to actin protein levels (N=4), (mean ± SD). Histogram and density plot representations of GPA⁺ CD71⁺ cells showing the levels of protein expression of FOXO3a and GATA1 in (E) HD-010 and (F) SS-010. (G) Western blot analysis of NRF2 (upper panel), FOXO3a (middle pane) and actin (lower panel) in nuclear extracts of differentiating HD and SS cells at D6 and D8 of differentiation. (H) Bar graph showing the quantification of FOXO3a (left) and NRF2 (right) protein levels in nuclear extracts from HD and SS cells at D6 and D8 of differentiation, relative to actin protein levels (N=4), (mean ± SD). *P<0.05, Mann-Whitney test.

Figure 3.

Erythropoiesis and oxidative stress heterozygous AS and homozygous SS Berkeley sickle mice. (A) Histogram representing Ter119 expression in the bone marrow (left) and spleen (right) cells of 1 AS (green) and 1 sickle cell anemia (SS) (blue) mouse. (B) Bar graph representing the percentage of Ter119⁺ cells in the bone marrow and the spleen of AS (N=5) and SS (N=8) mice, (mean ± standard deviation). (C) A bar graph representing the percentage of basophilic erythroblasts (EryA), polychromatic Ery (ryB) and orthochromatic Ery (EryC) cells in the bone marrow of AS and SS mice (N=7). (D) Bar graph representing the percentage of EryA, EryB and EryC cells in the spleen of AS and SS mice (N=7). (E) Histogram representing the levels of reactive oxygen species (ROS) in the peripheral blood of one AS (green) and one SS (blue) mouse. (F) Bar graph showing ROS mean fluorescence intensity (MFI) in the peripheral blood of AS (N=9) and SS (N=4) mice. (G) A bar graph representing ROS MFI in the bone marrow and spleen of AS and SS mice (N=5) (mean ± standard deviation). *P<0.05, **P<0.01, ***P<0.001 Mann-Whitney test.

Figure 4.

Ex vivo erythroid differentiation of Lin-negative cells from the bone marrow of AS and homozygous SS Berkeley mice.

Figure 5.

GATA1 protein levels in bone marrow and spleen cells of homozygous SS and heterozygous AS Berkeley mice. (A) Western blot images of GATA1 (upper panel) and actin (lower panel) proteins in nuclear extracts from Ter119⁺ cells, isolated from AS and sickle cell anemia (SS) bone marrow aspirates. (B) As in (A) using nuclear extracts from Ter119⁺ cells isolated from AS and SS spleens. (C) Bar graph representing quantification of GATA1 protein levels in the nuclear extracts of Ter119⁺ cells isolated from AS and SS bone marrow aspirates, relative to actin levels (N=2). (D) Bar graph representing quantification of GATA1 protein levels in the nuclear extracts of Ter119⁺ cells isolated from AS and SS spleens, relative to actin levels (N=4). (E) Bar graph representing the mean fluorescence intensity (MFI) of GATA1 in the Ter119⁺ population of the bone marrow (N=4) and spleen (N=4) of AS and SS mice (mean ± standard deviation). *P<0.05 Mann-Whitney test.

Figure 6.

GATA1 protein levels in bone marrow and spleen cells of homozygous SS and heterozygous AS Berkeley mice.(A) Imaging flow cytometry (IFC) analysis graph representing the Max Pixel_MC_Ch02 GATA1 FITC on the x-axis and the Raw Max Pixel_MC_ Ch02 GATA1 FITC on the y-axis. This analysis enables the discrimination between stage-matched GATA1-low erythroblasts and GATA1-high erythroblasts, within the Ter119⁺ erythroblast cell population. (B) Histogram representing the distribution of GATA1 staining in the GATA1-low erythroblasts (light blue) and GATA1-high erythroblasts (dark blue). (C) Bar graph representing the percentage of GATA1-high erythroblasts in the bone marrow (N=4) and spleen (N=3) of AS and SS mice (mean ± standard deviation). (D) Representative images of a GATA1-low erythroblast (upper panel) and a GATA1-high erythroblast (lower panel); images show Bright field, Ter119 (red), GATA1 (green) and the merged image. *P<0.05 Mann-Whitney test.

Figure 7.

Caspase 1 activity. Bar graph representing the percentage of FLICA-positive (caspase 1 active) cells in (A) the bone marrow and (B) spleen of AS and sickle cell anemia (SS) mice. The percentage of FLICA-positive cells is quantified in the basophilic erythroblasts (EryA, bone marrow), polychromatic Ery (EryB, bone marrow and spleen) and orthochromatic Ery (EryC, bone marrow and spleen) subpopulations (N=7). (C) Left panel: histogram showing FLICA levels in Ter119⁺ erythroblasts on day 2 of ex vivo differentiated Lin^- cells from the bone marrow of 1 AS and 1 SS mouse; right panel: bar graph representing the % FLICA-positive Ter119⁺ cells on day 2 of ex vivo differentiated Lin^- cells from the bone marrow of 1 AS and 1 SS mouse (N=2), (mean ± standard deviation). *P<0.05, **P<0.01, Mann-Whitney test.

Figure 8.

Inhibition of caspase 1 activity. (A) A contour plot of glycophorin A-positive (GPA⁺) cells from phase 2 of differentiation of sickle cell anemia (SS) untreated (dark blue) and SS treated with Ac-YVAD-cmk (50 µM) (light blue) CD34⁺ samples, showing the distribution of cell populations with respect to the expression of Band-3 FITC (x-axis) and CD49d-APC (y-axis). Data are represented for day 6 (D6) and D8 of phase 2 of differentiation. Pro: proerythroblast; EB: early basophilic; LB: late basophilic; Poly: polychromatic; Ortho: orthochromatic erythroblasts. (B) Images of SS untreated and Ac-YVAD-cmk treated cells at D8 of culture stained with MGG. (C) A histogram showing the analysis by flow cytometry of GPA⁺ cells, assaying for GATA1 protein on D6 and D8 of differentiation of SS untreated (dark blue) and SS Ac-YVAD-cmk treated (light blue) cells. (D) Bar graph representing the fold change in mean fluorescence intensity (MFI) of GATA1 in the GPA⁺ population at D6 and D8 of SS untreated (dark blue) and SS Ac-YVAD-cmk treated (light blue) cells during differentiation (N=3) (mean ± standard deviation). **P<0.01 Mann-Whitney.