Exportin 4, a Potential Amplifier of TGF-β Signaling, Is Increased in Primary Baboon Bone Marrow Erythroblasts Following Decitabine Treatment.

Abstract The DNA demethylating drug decitabine increased fetal hemoglobin (HbF) to therapeutic levels and reduced the level of DNA methylation of the γ-globin gene promoter in patients with sickle cell disease and in experimental baboons. Whether decreased DNA methylation of the γ-globin gene is solely responsible for increased HbF following decitabine treatment is unknown. Increased platelet counts in patients with sickle cell disease and myelodysplastic syndrome and in experimental baboons following decitabine treatment suggest that decitabine also affects hematopoietic differentiation. To investigate to what extent the mechanism responsible for the ability of decitabine to reactivate HbF and alter hematopoietic differentiation may involve the induction of other unknown genes, the global pattern of gene expression in purified primary bone marrow erythroblasts pre- and post-decitabine treament was analyzed. Baboons were phlebotomized for ten days (Hct 20) followed by adminstration of decitabine for ten days (0.52mg/kg/d; sc). RNA was isolated from nucleated erythroblasts purified from bone marrow aspirates obtained pre- and post-decitabine treatment. Purification of erythroblasts was performed by sedimentation in Percoll gradients followed by immunomagnetic column purification using an anti-baboon RBC antibody (Pharmingen). To assess the feasibility of using human Genechip arrays to detect differences in expression of baboon transcripts, RNA isolated from purified erythroblasts of a single baboon pre- and post-decitabine was hybridized in triplicate to human Genechip Focus arrays (Affymetrix) containing over 8500 genes. The expression of 48 genes was increased >2 fold in the post-decitabine treated sample compared to the pre-treatment sample. Among the more highly induced genes were HLA-A (3 fold), HLA-B (5.7 fold), exportin 4 (3.9 fold), and splicing factor 3b1 (3.7 fold). Reverse transcriptase PCR using human primer sets was performed to analyze the expression of these genes in pre- and post-decitabine treated bone marrow erythroblasts in independent samples from three additional baboons. Induction of HLA-A, exportin 4, and splicing factor 3b1 was confirmed in all three post-decitabine treated samples. The exportin 4 gene encodes a protein involved in nuclear export of the Smad3 protein. Activated TGF-β receptors phosphorylate Smad3 and induce its nuclear import to affect gene transcription. Following dephosphorylation of Smad3 in the nucleus, transport of the protein to the cytoplasm mediated by exportin 4 has been proposed to allow the propagation of multiple rounds of activation by activated TGF-β receptors thus amplifying TGF-β signaling (Kurisaki et al; Mol Cell Biol 26:1318, 2006). Because TGF-β increases HbF synthesis in cultured erythroid progenitors and also induces erythroid and megakaryocytic differentiation, we suggest that induction of exportin 4 by decitabine may play a role in the ability of this drug to increase HbF synthesis and alter hematopoietic differentiation. Our results thus confirm the feasibility of using human Genechip arrays to assess gene expression levels in baboons. Furthermore, we have indentified a gene induced by decitabine that potentially amplifies TGf-β signaling and thus may play a role in the ability of this drug to increase HbF and alter hematopoietic differentiation.