C/EBPβ Isoforms Regulate Proliferation and Differentiation of Regenerating Hematopoietic Stem/Progenitor Cells

Under stress or regenerative conditions, HSCs rapidly enter into cell cycle and are reprogrammed toward myeloid-biased hematopoiesis to meet the increasing demand of myeloid cells. We have previously shown that the transcription factor C/EBPβ plays critical roles at the level of HSPCs under stress conditions (Nat Immunol 2006, J Immunol 2012, Leukemia 2013 and Blood Adv 2019). However, underlying molecular mechanisms of action remain largely unknown. In this study, we have investigated the detailed function of C/EBPβ in regulation of HSPCs.

We first evaluated the impact of C/EBPβ on the cell cycle status of LT-HSCs. To exclude the cell-extrinsic contribution of C/EBPβ, CD45.2⁺ BM cells from WT or Cebpb^-/- mice were transplanted into lethally irradiated CD45.1⁺ WT mice, and these "BM-replaced" recipients were subjected to the following experiments. At steady state, the cell cycle statuses and the numbers of HSPCs did not significantly differ between the recipients of WT cells and those of Cebpb^-/- cells. Immediately after 5-FU treatment, WT LT-HSCs entered the cell cycle, as revealed by the decreased percentage of cells in G₀ phase and the increased percentage of cells in S/G₂M phase. All these parameters of cell cycle acceleration were observed prior to the nadir of LT-HSCs induced by 5-FU and were significantly attenuated in Cebpb^-/- LT-HSCs.

Next, we assessed the numbers of LT-HSCs, KSL cells, and KL cells after 5-FU treatment. Following the nadir, the recovery of LT-HSCs preceded that of KSL and KL cells, suggesting the differentiation of LT-HSCs to KSL and KL cells. In the recipients of Cebpb^-/- cells, the recovery of KSL and KL cells was delayed significantly. Collectively, cell cycle acceleration and subsequent differentiation of LT-HSCs under stress conditions were impaired in the absence of Cebpb.

The Cebpb is a single exon gene, and three isoforms, namely, LAP*, LAP and LIP which lacks N-terminus, are translated from its unique mRNA. Due to their structural difference, they should have distinct functions. Here, we focused on expression and functions of these isoforms in regenerating HSPCs. To monitor expression of these isoforms in small numbers of HSCs, we devised a novel intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies. An antibody against the C-terminus of C/EBPβ recognized all three isoforms, while an antibody against the N-terminus of C/EBPβ only recognized LAP* and LAP. Thus, simultaneous staining with both antibodies should enable us to distinguish cells that dominantly expressed LIP (C-term⁺ N-term^-) from those that expressed all three isoforms (C-term⁺ N-term⁺).

Using this method, we monitored the expression patterns of these isoforms in LT-HSCs after 5-FU treatment. LT-HSCs initially became C-term single positive in response to 5-FU and subsequently changed to C- and N-term double positive, suggesting that LIP was upregulated prior to LAP/LAP* under stress conditions. These results suggest that phase-specific upregulation of LIP and LAP/LAP* is strongly associated with phase-specific functions of C/EBPβ in cell cycle activation and differentiation, respectively. Indeed, when EML cells, a mouse HSC line, were retrovirally transduced with LIP, the transduced cells were more proliferative and actively cycling than those transduced with the control vector, whereas proliferation and cell cycle were markedly suppressed in LAP*- and LAP-expressing EML cells. LIP-expressing cells remained undifferentiated, while LAP*- and LAP-expressing cells rapidly differentiated into CD11b⁺ myeloid cells and eventually stopped proliferating.

In summary, our findings clearly suggest that sequential upregulation of C/EBPβ isoforms is critical for the regulation of HSCs under stress conditions. LIP amplifies the "reservoir" of HSPCs by accelerating the proliferation of HSCs during the early phase of regeneration, while LAP*/LAP induce their myeloid differentiation at a later phase. These findings should facilitate our understanding of the pathophysiology of infection, inflammation, and regenerating hematopoiesis in response to myeloablative chemotherapies or hematopoietic stem cell transplantation, all of which increase the hematopoietic demands.