Kras Plays An Important Role In Generating Differentiated Blood Cells

Kras is a small GTPase essential for mouse embryonic development. Although Kras^-/- fetal liver cells reconstitute recipient mice indistinguishably from wild-type cells, chimeric mice generated from injection of Kras^-/- embryonic stem cells into wild-type blastocysts show little contribution of knockout cells to hematopoietic tissues even when these cells contribute to all the other tissues to a high degree. These results suggest that Kras plays an important role in adult hematopoiesis. However, early embryonic lethality of Kras^-/- mice prevents further investigation of Kras functions in adulthood. To overcome this problem, we generated Kras conditional knockout mice (Kras^fl/fl), which allow the deletion of Kras by the Cre recombinase in desired tissues and at desired developmental stages.

We used two transgenic Cre lines, Mx1-Cre and Vav-Cre, to knockout Kras in adult hematopoietic system. The Mx1 promoter is induced by interferon signaling, which can be triggered by injections of polyinosinic-polycytidylic acid (pI-pC). The Vav promoter drives Cre expression specifically in fetal liver hematopoietic cells since E11.5 as well as in adult hematopoietic tissues. Both Cre lines efficiently deleted Kras expression in above 95% of hematopoietic cells as judged by single hematopoietic stem cell (HSC) genotyping. Results obtained from these two different Cre lines were essentially same.

We found that the frequency and absolute number of Kras^-/- HSCs, multipotent progenitors (MPPs), LSK (Lin^- Sca1⁺ cKit⁺) cells, myeloid progenitors and common lymphoid progenitors are comparable to wild-type control cells. Consistent with this observation, cytokine signaling in Kras^-/- hematopoietic stem/progenitor cells (HSPCs, Lin^- cKit⁺) is indistinguishable from control HSPCs. In contrast, the percentage of CD19⁺ B-cells is moderately but significantly reduced in Kras^-/- spleens and concomitantly cytokine-evoked ERK1/2 activation is greatly reduced in differentiated blood cells.

To determine whether Kras plays an important role in regulating HSC functions, we performed a competitive bone marrow reconstitution assay using CD45.2⁺ control or Kras^-/- bone marrow cells mixed together at ratios 1:1 and 3:1 with congeneic competitor cells (CD45.1⁺ bone marrow cells). Kras^-/- bone marrow cells show significantly reduced long-term reconstitution in recipient mice compared to control cells (10% vs 45%). The reduced reconstitution is persistent in the secondary and tertiary recipients. However, detailed analysis in primary and secondary recipients revealed that the frequency of Kras^-/- HSCs and MPPs is comparable to that of control cells and Kras^-/- progenitor cells are also largely normal, indicating that Kras is dispensable for adult HSC functions but might play an important role in generating differentiated blood cells. The reduced generation of myeloid cells is further validated in an in vitro culture assay, in which we quantitatively measured the myeloid cell production from Lin^- progenitor cells.

Our results indicate that loss-of-Kras could be compensated by other Ras isoforms in adult HSCs. However, in mature blood cells, Kras deficiency results in greatly reduced cytokine-evoked ERK1/2 activation. Under a stressed condition (e.g. competitive bone marrow transplantation), the generation of Kras^-/- blood cells is defective. Taken together, our study reveals a novel and unique function of Kras in regulating adult hematopoiesis.

No relevant conflicts of interest to declare.