Multiple physical stresses induce γ-globin gene expression and fetal hemoglobin production in erythroid cells

Increased fetal hemoglobin (HbF) expression is beneficial for β-hemoglobinopathy patients; however, current inducing agents do not possess the ideal combination of efficacy, safety and ease of use. Better understanding the mechanisms involved in γ-globin gene induction is critical for designing improved therapies, as no complete mechanism for any inducing agent has been identified. Given the cytotoxic nature of most known inducing drugs, we hypothesized that γ-globin is a cell stress response gene, and that induction occurs via activation of cell stress signaling pathways. We tested this hypothesis by investigating the ability of physical stresses including heat-shock (HS), UV- and X-irradiation and osmotic shock to increase γ-globin gene expression in erythroid cells. Experiments in K562 and KU812 cells showed that each of these stresses increased steady-state γ-globin mRNA levels, but only after 3-5days of treatments. HS and UV also increased γ-globin mRNA and HbF levels in differentiating primary human erythroid cells. Mechanistic studies showed that HS affects γ-globin mRNA at multiple levels, including nascent transcription and transcript stability, and that induction is dependent on neither the master regulator of the canonical HS response, HSF1, nor p38 MAPK. Inhibitor panel testing identified PI3K inhibitor LY294002 as a novel inducing agent and revealed potential roles for NFκB and VEGFR/PDGFR/Raf kinases in HS-mediated γ-globin gene induction. These findings suggest that cell stress signaling pathways play an important role in γ-globin gene induction and may provide novel targets for the pharmacologic induction of fetal hemoglobin.