Hydroxyurea (HU) has been shown to augment the production of fetal hemoglobin (HbF) and for this reason is being used in the treatment of sickle cell anemia.It has been assumed that hydroxyurea (HU) promotes HbF production indirectly by perturbing the maturation of erythroid precursors. However, the molecular mechanism(s) involved in how HU regulates gamma-globin expression remains unclear. Using a two-phase liquid erythroid culture system in conjunction with mRNA differential display, we reported the cloning of a HU-inducible small GTP-binding protein, designated SAR-e. SAR-e is localized to chromosome 10 and is expressed in most tissues with high level of expression in thyroid, spinal cord and adrenal gland and low level in adult bone marrow. SAR-e tagged with a GFP fusion protein at the 3' end co-expressed with anti-calreticulin in the ER complex in K562. We stably transfected the SAR-e gene into K562 cells and found a 2.4 fold induction of g-globin gene expression determined by Northern Blot. Enforced expression of SAR-e leads to a striking macrocytosis and a more immature appearance of the erythroid cells, with a doubling time of 30 hours compared with 18 hours for wild-type K562 cells. By flow cytometric cell cycle analysis, the percentage of cells in G2/M phase was decreased from 26% (wild-type) to 6% in transfected K562 cells. This inhibition of cell growth and differentiation, apoptosis and accumulation of cells in the S-phase are consistent with the known effects of HU on erythroid cells. Recent data strongly indicates a tight correlation between SAR and gamma-globin gene expression which was demonstrated by the following different set experiments: the correlation coefficient between SAR and gamma-globin transcriptional level is 0.74 in SAR stable transfected K562 cells; parental K562 cells were treated with various hemoglobin gene expression inducers (HU, butyrate, and 5-azacytidine) for 48hrs respectively, Northern blot displayed these inducers upregulated ?-globin mRNA from 2.54-fold for butyrate to 2.95-fold for HU along with paralleled increase of SAR mRNA from 1.54-fold for butyrate to 1.98-fold for HU; SAR and ?-globin expression in the hematopoietic stem/progenitor cell line, AC133+, cultured with EPO were analyzed by RT-PCR at different time points; the result showed both SAR and gamma-globin expression began to be upregulated at day 10, reached the peak at day 21 and then diminished at day 28, mirroring the gene expression time course patterns seen in vivo. These recent experiments experiments suggests that the SAR gene might be a common mediator of ?-globin gene expression. Western blots showed phosphorylated extracellular signal-regulated kinases (ERK) were decreased 79% in stably tranfected cells compared to vector-only cells. GATA-1 protein was down regulated and GATA-2 was up regulated by SAR-e. Our data supports a novel role for the small GTP-binding protein distinct for its known protein trafficking function, involving both cell cycle control and gene expression regulation in erythroid cells. Further elucidation of the involved pathways utilizing siRNA and specific protein trafficking inhibitors, as well as an analysis of the function of retroviral delivered SAR into primary hematopoietic cell are currently underway.
| Tang, Delia C; Zhu, Jianqiong; Liu, Wenli et al. (2005) The hydroxyurea-induced small GTP-binding protein SAR modulates gamma-globin gene expression in human erythroid cells. Blood 106:3256-63 |
