The Role of Reactive Oxygen Species in γ-Globin Gene Activation by Fetal Hemoglobin Inducers.

Enhancing γ-globin expression is an effective therapeutic strategy for sickle cell disease. Fetal hemoglobin (HbF) inducers such as sodium butyrate (NaB), trichostatin A (TSA) and hydroxyurea (HU) have been studied extensively to ascertain molecular and cell signaling mechanisms for γ-gene activation. Reactive oxygen species (ROS) including nitric oxide, superoxide, hydroxyl radicals and hydrogen peroxide stimulate signaling through the cyclic guanosine monophosphate (cGMP) and p38 MAPK pathways. Indirect evidence supports HU as a nitric oxide donor thus providing a mechanism for HbF induction via cGMP activation. Recently, we and others demonstrated a role for p38 signaling in γ-gene induction by HU as well as NaB, TSA and apicidin. However, the ability of HbF inducers to stimulate ROS formation as a mechanism for γ-globin activation via cGMP or p38 MAPK signaling has not been determined. To clarify this mechanism we measured ROS levels using 2′,7′-dichlorofluorescin diacetate which emits fluorescence at a 529 nm wavelength when activated by hydrogen peroxide. We correlated ROS levels with p38 phosphorylation and γ-globin mRNA levels measured by western blot and real-time PCR respectively. Studies were also completed in the absence and presence of myxothiazol, a known inhibitor of ROS formation to confirm our findings. The positive controls menadione and hemin increased ROS levels 4-fold and 3-fold with a concomitant 6.5-fold and 4-fold increase in γ-globin mRNA respectively. Pretreatment with myxothiazol inhibited maximal HbF induction by both agents. By contrast, NaB and TSA did not increase ROS levels in K562 cells therefore they activate γ-globin by a ROS-independent, p38 MAPK-dependent mechanism. We concluded that ROS play a major role in the mechanism for HbF induction by hemin and the vitamin K derivative, menadione. Investigations were completed which confirmed the ability of HU to stimulate ROS formation as well. Studies are underway to determine the role of hydrogen peroxide and/or nitric oxide in HbF induction by HU via cGMP/p38 MAPK cell signaling.