The HU Inducible SAR1 Gene Acts Through GαI /JNK/JUN Pathway To Activate γ-Globin Expression

Hydroxyurea (HU) has been effectively used in the treatment of β-hemoglobinopathies by augmenting γ-globin chain to compensate for the impaired or defective β-globin chain, but the molecular mechanism underlying HU-mediated γ-globin regulation remains unclear. We previously reported that over-expression of the HU-induced SAR1 gene increased γ-globin expression in primary erythroid cells, and SAR1 is required for HU's effects in this process. Here we present further mechanistic details underlying SAR1’s effects. First, we examined the role of COPII pathway in HbF expression by genetically silencing the key downstream COPII components Sec12/Sec23 or by chemical inhibition of vesicle protein transport from ER to Golgi with BFA. We found that blocking this pathway through siRNA Sec12/23 or BFA only reduced HU-induced γ-globin expression by 32% and 28%, respectively, without a change in basal HbF level (as determined by QT-PCR or flow cytometry). These data suggested that other signaling pathways might account for HU- and SAR1-mediated HbF induction. Additional studies also indicated that stable tranfected SAR1 was able to activate Gαi /JNK/JUN signaling in erythroid cell. Inhibition of JNK/JUN activation by the JNK inhibitor SP600125 was able to reduce by 8.79% and 38.63% HbF positive cells in vector- and SAR1-transfected-cells, respectively. Silencing of Gαi2 or Gαi3, but not Gαi1 or Gαq expression in SAR1-stable transfected cells could significantly eliminate SAR1-stimulated JUN protein phosphorylation, and resulted in the 54% and 46% reduction of γ-globin expression, respectively. Reciprocal co-immunoprecipitation assays revealed a direct association between Sar1 and Gα (Gαi and Gαq) protein in both erythroid and non-erythroid cells. These data taken together with our previous findings indicate that HU induces SAR1 which in turn activates γ-globin expression predominantly through the Gαi/JNK/JUN pathway. These findings illuminate an alternative approach to targeted therapy for the beta-globin disorders.