| Increased fetal hemoglobin (HbF) expression is beneficial for beta-hemoglobinopathy patients; however, most inducing agents do not possess the ideal combination of efficacy, safety and availability. Better understanding the mechanisms involved in gamma-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 inducers, we hypothesized that gamma-globin is a cell stress response gene, and that induction occurs via activation of cell stress signalling pathways. We tested this hypothesis by investigating the ability of physical stresses including heat-shock (HS), UV, X-irradiation and osmotic shock to increase gamma-globin gene expression in erythroid cells. Experiments in K562 and KU812 cells showed that each of these stresses increased steady-state gamma-globin mRNA levels, but only after 3-5 days of treatments. HS and UV also increased gamma-globin mRNA and HbF levels in differentiating primary human erythroid cells. Mechanistic studies showed that HS affects gamma-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. Past literature reports and findings from our laboratory prompted us to initially place p38 at the centre of our model; therefore we also investigated the effect of p38 inhibitor SB203580 (SB) on gamma-globin gene expression. SB dose-dependently decreases basal gamma-globin mRNA levels; however, genetic modulation of p38 did not affect basal or HS-induced gamma-globin gene expression, but did attenuate sodium butyrate (NaB)-mediated increases. In comparing the effects of SB to an alternative p38 inhibitor, BIRB0796, we identified a role for SB in gamma-globin gene regulation that is partially independent of its canonical function as a p38 inhibitor. Preliminary data suggests that SB-mediated ERK activation may be involved in this gamma-globin suppression. Additionally, testing of a panel of inhibitors revealed a potential role for NFkappaB in stress-mediated gamma-globin gene induction and identified PI3K inhibitor LY294002 as a novel inducing agent. These findings suggest that cell stress signalling pathways play an important role in gamma-globin gene induction, and may provide novel targets for the pharmacologic induction of fetal hemoglobin. |
