The effect of histone deacetylase inhibitors on AHSP expression

Abstract

Alpha-hemoglobin stabilizing protein (AHSP) is a molecular chaperone that can reduce the damage caused by excess free α-globin to erythroid cells in patients with impaired β-globin chain synthesis. We assessed the effect of sodium phenylbutyrate and sodium valproate, two histone deacetylase inhibitors (HDIs) that are being studied for the treatment of hemoglobinopathies, on the expression of AHSP, BCL11A (all isoforms), γ-globin genes (HBG1/2), and some related transcription factors including GATA1, NFE2, EKLF, KLF4, and STAT3. For this purpose, the K562 cell line was cultured for 2, 4, and 6 days in the presence and absence of sodium phenylbutyrate and sodium valproate. Relative real-time qRT-PCR analysis of mRNA levels was performed to determine the effects of the two compounds on gene expression. Expression of all target mRNAs increased significantly (p < 0.05), except for the expression of BCL11A, which was down-regulated (p < 0.05) in the cells treated with both compounds relative to the levels measured for untreated cells. The findings indicated that sodium valproate had a more considerable effect than sodium phenylbutyrate (p < 0.0005) on BCL11A repression and the up-regulation of other studied genes. γ-Globin and AHSP gene expression continuously increased during the culture period in the treated cells, with the highest gene expression observed for 1 mM sodium valproate after 6 days. Both compounds repressed the expression of BCL11A (-XL, -L, -S) and up-regulated GATA1, NFE2, EKLF, KLF4, STAT3, AHSP, and γ-globin genes expression. Moreover, sodium valproate showed a stronger effect on repressing BCL11A and escalating the expression of other target genes. The findings of this in vitro experiment could be considered in selecting drugs for clinical use in patients with β-hemoglobinopathies.

Fig 1. The effect of sodium phenylbutyrate and sodium valproate on the expression of AHSP, BCL11A, γ-globin genes (HBG 1/2) and erythroid transcription factors.

Both compounds led to significant induction of AHSP, γ-globin genes (HBG1/2) and erythroid transcription factors in K562 cells (p < 0.05). However, BCL11A was significantly repressed due to treatment with them (p < 0.05). Higher concentrations of the studied HDIs and longer treatment times led to significantly greater repression of BCL11A and higher expression of other target genes (p < 0.0005). NB, sodium phenylbutyrate; NV, sodium valproate; 2D, two days of treatment; 4D, four days of treatment; 6D, six days of treatment.

Fig 2. Comparison of the effect of sodium phenylbutyrate and sodium valproate on the expression of AHSP, BCL11A, γ-globin genes (HBG 1/2) and erythroid transcription factors after six days of treatment.

Sodium valproate was significantly more efficient on the down-regulation of BCL11A and the up-regulation of AHSP, γ-globin genes (HBG 1/2), and erythroid transcription factors compared with sodium phenylbutyrate (p < 0.0005). NB, sodium phenylbutyrate; NV, sodium valproate.

Fig 3. Protein expression analysis by western blotting.

K562 cells were cultured with or without sodium phenylbutyrate 1 mM (NB), sodium valproate 1 mM (NV) and hemin 50 μM. After 6 days, the cells were examined for STAT3, AHSP, γ-globin and BCL11A protein expression by Western blot analysis. To normalize protein loading, the blot was hybridized with β-actin antibody.