Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways

Abstract

When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D)) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.

Figure 1

Expression of oncogenic K-ras can be efficiently induced from its endogenous promoter by a Tet-on system. (A) Schematic representation of the tetracycline-inducible system, which involves in breeding 3 lines together. The first line is the M2rtTA transactivator driven from the endogenous Rosa26 promoter (R26-M2rtTA). The second line contains 2 alleles, cre recombinase driven by the Tet operator (LC-1) and a floxed β-galactosidase-neo (βgeo) reporter cassette at the endogenous Rosa26 locus (R26R). The third line is the conditional oncogenic K-ras at its endogenous locus. (B) TER119-negative cells were purified from individual embryos that carried all the alleles and were induced with or without doxycycline (Dox) for 42 to 48 hours. βGeo activity was detected by a fluorogenic substrate fluorescein di-β-D-galactopyranoside (FDG).

Figure 2

Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of Epo-dependent signaling pathways. At E15.5, fetal liver cells were isolated from individual embryos that had been induced with doxycycline for the previous 42 to 48 hours. Cells were simultaneously stained for CD71 and TER119, and erythroid differentiation was analyzed (A). The percentages of R1 + R2 cells (predominantly primitive progenitor cells, including mature BFU-Es and CFU-Es) are shown in brackets on individual graphs (A) and further quantified (error bars represent standard deviations) (B). (C) TER119-negative cells were purified from individual E15.5 embryos, deprived of serum and growth factors for 30 minutes, then stimulated with various concentrations of Epo for 10 minutes. Phosphorylated and total levels of Stat5, p44/42 ERK, and Akt proteins were measured by Western blotting (see “Materials and methods”).