55. Alteration of Primitive Hematopoietic Cell Fate and Growth by the GATA-2 Transcription Factor

Abstract

The GATA-2 transcription factor is required for the development and regulation of blood stem cells as judged by the lethal, “bloodless” phenotype of mice lacking GATA-2 and the high levels of GATA-2 present in adult stem cells which necessarily decline with differentiation into the various blood lineages. This idea is consistent with our previous studies showing that sustained, high level GATA-2 expression in adult murine stem cells blocks both stem cell amplification and differentiation in vivo (Persons, Blood 93:488, 1999). Recent studies implicate GATA-2 in maintaining p21 and p27 proteins (Ezoe, Blood 100:3512,2002), thereby contributing to the quiescence of the stem cell pool. In order to determine if p21 and p27 mediate this role in hematopoietic stem cells (HSCs) in vivo, a GATA-2 retroviral vector was used to enforce expression in p21/p27 null HSCs. Although Sca+Lin− p21/p27 null cells were efficiently transduced with both the GATA-2 and control vectors, engraftment of transduced cells was only observed with the control vector. These data suggest that GATA-2 regulated stem cell quiescence is independent of p21/p27. To further investigate the stem cell regulatory activity of GATA-2, we developed a retroviral vector encoding a GATA-2/estrogen receptor chimeric protein (G2-ERT2). Hematopoietic progenitors transduced with the retroviral G2-ERT2/GFP vector generated few GFP+ colonies in the presence of tamoxifen, relative to the number formed in the absence of tamoxifen (6 ± 5%, n = 5), consistent with our previous findings. In the absence of tamoxifen, G2-ERT2 colonies grew significantly larger in size. In addition, G2-ERT2 transduced bone marrow (BM) cells out-competed mock transduced cells in liquid culture. Control GFP or G2-ERT2 transduced total BM cells were diluted with mock transduced BM cells at a 1:10 ratio. At 7 days, the control culture was 10% GFP+ and the G2-ERT2 culture was 17% positive. However, by day 11 the control culture remained 10% GFP+, while the G2-ERT2 culture increased to 87% GFP+. In contrast to control cultures, the G2-ERT2 cultures were predominantly composed of immature-appearing cells. This G2-ERT2 pro-proliferative effect was also observed in vivo. We found a significant repopulation advantage of G2-ERT2 myelo-erythroid cells over untransduced cells in mice transplanted with G2-ERT2 transduced BM. Mice transplanted with G2-ERT2 transduced BM mixed 1:1 with untransduced marrow at 12 weeks showed 82% ± 6 GFP+ Gr-1+ cells (n = 10) and 57% ± 4 RBCs compared to 28% ± 17 (n = 7) and 10% ± 5 in GFP:Mock control mice. Interestingly, there was a lack of lymphoid development since few B220+ cells co-expressed GFP in G2-ERT2 transplanted mice at 12 weeks (8% ± 2), compared to controls (58% ± 14). Since it is likely that small amounts of G2-ERT2 are active in the absence of tamoxifen, the concentration of GATA-2 is likely critical in normally regulating primitive hematopoietic cell function and fate decisions. Thus, constitutive, low concentrations of GATA-2 lead to a pro-proliferative effect in myeloid cells but inhibit lymphoid development. Since our data suggest that GATA-2 does not mediate its effects through the p21/p27 pathway, investigation of GATA-2 interactions with other transcription factors and their effects on gene regulation is needed.