mTOR-C/EBPβ-c-Myc Axis Regulates the Behavior of Myeloid-Biased Hematopoietic Stem Cells Under Stress Conditions

In stress or regenerative conditions, hematopoietic stem cell (HSC) population drastically alters its behavior; they rapidly enter into cell cycle, differentiate to multipotent progenitors (MPPs) at the expense of self-renewal activity, and are reprogrammed toward predominantly myeloid-biased hematopoiesis. In these processes, essential roles of myeloid transcription factors in regenerating HSCs are anticipated. C/EBPβ regulates both proliferation and differentiation of granulocytic precursors in stress conditions. However, its functions in HSCs during stresses are largely unknown.

In the past several years, we have been investigating the roles of C/EBPβ in HSCs under stress conditions, especially focusing on the expression pattern of C/EBPβ isoforms and their respective functions. Cebpb is a unique single exon gene, from which two long protein isoforms (LAP* and LAP) and one truncated isoform lacking N-terminal activating domain (LIP) are translated by different usage of initiating codons. To monitor the expression pattern of these isoforms in scarce cells, we have devised an intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies recognizing N- and C-terminus, respectively (57th ASH annual meeting, #3580).

Here, we refined this method to reveal the characteristic expression pattern of C/EBPβ isoforms in HSCs in vivo in response to stresses. In regenerative conditions after either 5-FU treatment or transplantation, we detected remarkable LIP-dominant upregulation in LT-HSCs at early phases, which was followed by increase of LAP*/LAP at later phases. This stress-induced translational upregulation and alteration of C/EBPβ were significantly suppressed by short-term administration of rapamycin, a selective inhibitor of mTOR, suggesting its dependence on mTORC1 pathway at least in part.

We further found that the stress-induced LIP-dominant upregulation of C/EBPβ was remarkable in LT-HSCs with higher surface CD150 expression (CD150^high LT-HSCs), which possess myeloid-biased potential. Cell cycle analyses at early phases after 5-FU treatment, when LIP was predominantly expressed, revealed that CD150^high HSCs were significantly less quiescent than CD150^low HSCs but this difference was abolished in Cebpb^-/- HSCs, suggesting that C/EBPβ specifically activated the cell cycle of CD150^high "myeloid-biased" HSCs.

We next examined the functions of C/EBPβ isoforms in HSCs in vivo . We transplanted WT BM cells retrovirally transduced with each C/EBPβ isoform into sublethally irradiated WT mice, and then analyzed the transduced cells in the recipients. LAP*- or LAP-transduced cells disappeared as early as 3 weeks after transplantation, probably due to their rapid myeloid differentiation. LIP-transduced HSCs were significantly less quiescent, exhibited more accelerated differentiation to MPPs, especially lymphoid-primed MPP4s, and produced more lymphoid-biased outputs. These results indicate that LIP induces cell cycle activation of HSCs and expansion of MPP pool, while antagonizing the other long isoforms that induce rapid myeloid commitment and premature exhaustion of HSCs.

As a candidate for the downstream target of C/EBPβ, especially LIP, in HSCs, we focused on Myc (encoding c-Myc). LIP-transduced EML cells, a mouse HSC line, expressed 1.5-fold higher level of Myc mRNA than those transduced with control, and LIP-transduced HSCs in vivo expressed 2-fold higher than control. We next compared Myc mRNA expression between WT and Cebpb^-/- HSCs at early phases of regeneration post 5-FU treatment and transplantation, when LIP was predominantly upregulated. Then, we found that significantly lower level of Myc transcripts was expressed in Cebpb^-/- HSCs. When we performed a ChIP-qPCR analysis using C/EBPβ-transduced EML cells, remarkable enrichment of C/EBPβ was observed at the distal enhancer of Myc gene. These data suggest that both forced overexpression and physiological elevation of LIP positively regulate Myc transcription in HSCs likely through direct binding to Myc enhancer.

In summary, our findings illustrate that stress-induced upregulation and alteration of C/EBPβ protein, mediated by mTORC1 and modulating Myc transcription, is significantly involved in stress-mode behavior of HSCs especially with myeloid bias.