Thalidomide induces gamma-globin gene expression through increased reactive oxygen species-mediated p38 MAPK signaling and histone H4 acetylation in adult erythropoiesis

Abstract

Although thalidomide has been shown to improve anemia in some patients with myelodysplastic syndromes and stimulates erythropoietin in patients with multiple myeloma, thalidomide's specific effects on gamma-globin gene expression during erythroid differentiation have not been studied. Here, we investigated the effects of thalidomide on gamma-globin gene expression and the involved signaling pathway using an ex vivo culture system of primary human CD34+ cells. We found that thalidomide induced gamma-globin mRNA expression in a dose-dependent manner, but had no effect on beta-globin expression. We also demonstrated that intracellular reactive oxygen species (ROS) levels were increased by treatment with thalidomide for 48 hours (from day 3 to day 5). Western blot analysis demonstrated that thalidomide activated the p38 mitogen-activated protein kinase (MAPK) signaling pathway in a time- and dose-dependent manner and increased histone H4 acetylation. Pretreatment of cells with the antioxidant enzyme catalase and the intracellular hydroxyl scavenger dimethylthiourea (DMTU) abrogated the thalidomide-induced p38 MAPK activation and histone H4 acetylation. Moreover, pretreatment with catalase and DMTU diminished thalidomide-induced gamma-globin gene expression. These data indicate that thalidomide induces increased expression of the gamma-globin gene via ROS-dependent activation of the p38 MAPK signaling pathway and histone H4 acetylation.

Figures

Figure 1

Effects of thalidomide on cell growth and erythroid colony formation. (A) Human CD34+ cells were cultured in erythropoietin (EPO) medium with the indicated concentration of thalidomide for 2 weeks and counted on day 14. (B) Erythroid colonies (BFU-E + CFU-E) were calculated as numbers of colonies per well. CD34+ cells (1 × 103 cells/well) were cultured in methylcellulose with the indicated concentration of thalidomide. CFU-Es (▩) were evaluated at day 7 and BFU-Es (■) were evaluated at day 14. (C) Thalidomide (1-100 μM) was added to CD34+ cell cultures either for only the first week (days 0-7) or the second week (days 7-14), and the cells were counted on day 7 or day 14. Results are shown as means (± SD) from 3 different donors. *P < .05 versus untreated cells.

Figure 2

Effects of thalidomide on expression of γ- and β-globin mRNA in erythroid progenitor cells. CD34+ cells were cultured for 14 days in EPO and treated with different concentrations of thalidomide (0.01-100 μM) over the indicated time periods (days 0-14, days 0-6, or days 6-14). (A,C) γ-globin and (B,D) β-globin mRNA levels were quantitated by real-time PCR. Results are shown as means (± SD) from 3 different donors. *P < .05 versus untreated cells.

Figure 3

Thalidomide induces activation of p38 MAPK and acetylation of histone H4. CD34+ cells were cultured in EPO alone medium for 7 days prior to the addition of thalidomide. (A) Cells were treated with different concentrations of thalidomide for 30 minutes. (B) Cells were treated with 100 μM thalidomide for the indicated times. Cellular protein (30 μg) was analyzed by Western blot using phosphorylated p38 MAPK and acetyl-histone H4 antibodies. The membranes were stripped and reprobed with total p38 MAPK and histone H4 antibodies as indicated to confirm that similar amounts of protein extracts were analyzed in each lane.

Figure 4

Thalidomide induces intracellular ROS in erythroid progenitor cells. CD34+ cells were treated with the indicated concentration of thalidomide (A) or with 100 μM thalidomide in the presence or absence of catalase (4000 U/mL), DMTU (10 mM), or SB203580 (5 μM) (B) from day 3 to day 5 for 48 hours, followed by treatment with the DCFDA probe for 10 minutes. The intracellular reactive oxygen species (ROS) production was measured by flow cytometry. ROS levels induced by thalidomide were normalized to those of untreated cells as determined by monitoring the increased fluorescence in the cells. Results are shown as means (± SD) from 3 independent experiments. *P < .05 versus untreated cells.

Figure 5

Increased p38 MAPK phosporylation and acetylation of histone H4 by thalidomide inhibited by the antioxidants catalase and DMTU, and p38 inhibitor SB203580. CD34+ cells cultured in EPO medium for 7 days were pretreated with the antioxidants catalase (4000 U/mL) or DMTU (10 mM) (A) or with 5, 10, or 20 μM of the p38 MAPK inhibitor SB203580 (B) for 30 minutes, then coincubated with 100 μM thalidomide in the presence of indicated inhibitors for 30 minutes. Cell lysates (30 μg) were analyzed by Western blot using phosphorylated p38 MAPK and acetyl-histone H4 antibodies. Total p38 MAPK and histone H4 were analyzed as loading controls as described in Figure 3.

Figure 6

Thalidomide-induced γ-globin expression requires enhanced ROS and mediated p38 MAPK activation. CD34+ cells grown in EPO medium for 6 days were treated with 100 μM thalidomide in the presence or absence of the antioxidants catalase (4000 U/mL) or DMTU (10 mM) or the p38 MAPK inhibitor SB203580 (5 μM) from days 6 to 14. RNA extracted from cells harvested on day 14 was amplified by quantitative PCR to determine γ- and β-globin expression levels. (A) Average copy number of γ-globin molecules per 1 ng total RNA. (B) Average copy number of β-globin molecules per 1 ng total RNA. (C) Average ratio of γ/γ + β-globin percentages. Results are shown as means (± SD) from 3 independent donors that were analyzed in separate experiments. *P < .05 versus untreated cells.

Figure 7

Thalidomide induced fetal hemoglobin expression. Cells were cultured in EPO on days 0 to 6 and then were treated with the indicated concentration of thalidomide (A) or 100 μM thalidomide in the presence or absence of catalase (4000 U/mL), DMTU (10 mM), or SB203580 (5 μM) (B) from days 6 to 14. On day 14, cells were stained with fluorescently labeled anti-HbF antibodies and analyzed by flow cytometry. Panels represent fluorescence signal (y-axis) versus cell size (x-axis); the percentage of positive cells is shown in the top right corner of each panel. The panels shown are representative of the results for cultures from 3 separate donors.