Delta-Aminolevulinate Preferentially Induces Gamma-Globin Expression in Erythroid Cells through Activation of NRF2 Stress Response

Fetal hemoglobin (HbF,α2γ2) is an important genetic modifier for β-hemoglobinopathies including sickle cell disease (SCD) and β-Thalassemia which occur as a result of mutations in the adult β-globin gene.  δ-aminolevulinate (ALA), the first product synthesized in the mammalian heme biosynthesis pathway, has been reported to induce hemoglobin production.  In this study, we determined the effects of ALA on γ- and β-globin transcription and HbF production in an erythroid cell line and primary erythroid progenitors.  We demonstrated that ALA increased heme levels by 50% with 48 hour treatment in KU812 erythroleukemia cells.  By reverse transcription-real time polymerase chain reaction (RT-qPCR), we detected an induction of γ-globin transcription in a time- and dose-dependent manner with 2.4-fold elevation at the 2mM ALA concentration.  Moreover, we showed that HbF protein level increased dramatically by 22.9-fold at 48 hour ALA treatment by western blot analysis with HbF specific antibody.  By contrast, ALA treatment did not change β-globin transcription level.  Subsequent studies in primary erythroid progenitors derived from CD34+ stem cells, we observed γ-globin transcriptional activation  up to 6.5-fold by day 22 in culture with 48hour ALA treatment, whereas β-globin transcription was not significantly changed.  Studies to determine molecular mechanisms demonstrated that addition of succinylacetone (SA), a specific inhibitor of heme biosynthesis, blocked the ALA-mediated induction of γ-globin transcription and HbF synthesis in KU812 cells detected by RT-qPCR, western blot and enzyme-linked immunosorbent assay.  Using 2’-7’-Dichlorodihydrofluorescein diacetate with flow cytometer detection, we observed a 3-fold increase of reactive oxygen species such as hydrogen peroxide with 24-hour ALA treatment in KU812 cells which was inhibited by the addition of SA.  In addition, western blot analysis detected increased nuclear translocation of NRF2, a master transcription factor controlling the cellular antioxidant response.  Using chromatin immunoprecipitation, we demonstrated that NRF2-associated enrichment of γ-globin proximal promoter chromatin was enhanced 10-fold in the region of the antioxidant responsive element.  Furthermore, in KU812 cells transiently transfected with a wild-type and ARE-mutated γ-globin promoter luciferase reporters followed by ALA treatment, we detected activation of the wild-type γ-globin promoter-driven luciferase reporter.  As predicted, deletion of the ARE in the γ-globin promoter not only significantly reduced the basal activity by 10-fold but also abolished the ALA-induced promoter activity.  These data support a role of ALA in activating heme production and γ-globin expression through an NRF2 activation mechanism.  Our results provide evidence that ALA and the heme biosynthesis pathway are novel targets for therapeutic potential in treating β-hemoglobinopathies.