Csf1r Is a Downstream Target of C/EBPβ in Ly6C¯ Monocytes

Currently, monocytes are classified into at least two subsets. Classical monocytes, also known as inflammatory monocytes (a Ly6C⁺ subset in mice and a CD14⁺ CD16⁻ subset in human), are involved in innate immune responses. On the other hand, patrolling monocytes (a Ly6C⁻ subset in mice and a CD14⁻ CD16⁺ subset in human) have been recently identified. Ly6C⁻ monocytes are found attached on the luminal side of endothelium and scavenge microparticles. Developmentally, Ly6C⁺ and Ly6C⁻ monocytes share common monocyte progenitors (cMoPs), or Ly6C⁻ monocytes might be converted from Ly6C⁺ monocytes. Although involvement of Ly6C⁻ monocytes in various kinds of diseases has been reported, molecular mechanisms which regulate the homeostasis of Ly6C⁻ monocytes are largely unknown.

CCAAT/Enhancer Binding Protein β (C/EBPβ) is a leucine zipper type transcription factor. We and others have previously shown that C/EBPβ is required for stress-induced granulopoiesis (Hirai et al. Nat Immunol, 2006, Satake et al. J Immunol, 2012, Hayashi et al. Leukemia 2013). However, its roles in steady state hematopoiesis remain relatively unknown. We have recently found that peripheral blood monocytes are significantly reduced in Cebpb^−/− mice (Tamura et al. Biochem Biophys Res Commun, 2015). In addition, last year in this meeting, we have reported that Cebpb mRNA is highly upregulated during differentiation from myeloid progenitors or Ly6C⁺ monocytes to Ly6C⁻ monocytes, and that Ly6C⁻ monocytes are almost completely absent in Cebpb^−/− mice due to enhanced cell death [Abstract #224]. Here, we further investigated the molecular mechanisms underlying C/EBPβ-dependent survival of Ly6C⁻monocytes.

In this study, we focused on the regulation of Csf1r (also known as M-CSF receptor). Csf1r is an essential molecule for the development and survival of monocytes. To determine the developmental stages at which Csf1r plays critical roles, we measured the expressions of Csf1r mRNA in hematopoietic stem/progenitor cells and monocyte subsets obtained from wild-type (WT) mice. Csf1r mRNA was expressed at at low levels in hematopoietic stem/progenitors including macrophage dendritic precursors (MDPs) and cMoPs. Csf1r mRNA started to be upregulated in Ly6C⁺ monocytes, followed by a drastic increase in Ly6C⁻ monocytes. These expression patterns were quite similar to those of Cebpb, suggesting the close relationship between Csf1r and C/EBPβ. Interestingly, such drastic increase of Csf1r mRNA in Ly6C⁻ monocytes was blunted in Cebpb^−/− mice, and protein levels of Csf1r in Cebpb^−/− Ly6C⁻ monocytes were significantly lower than those in WT Ly6C⁻ monocytes. In order to evaluate the effect of C/EBPβ overexpression on Csf1r expression, EML cells, a mouse hematopoietic stem cell line, were engineered to express C/EBPβ-estrogen receptor (ER) fusion protein or ER alone. Nuclear translocation of C/EBPβ-ER in the presence of tamoxifen resulted in significantly increased levels of Csf1r mRNA and protein when compared to nuclear translocation of ER alone. Previous reports have demonstrated that a combination of a promoter sequence and an enhancer region located in the first intron of Csf1r gene (Fms intronic regulatory element: FIRE) is enough to recapitulate the endogenous Csf1r expression and that these elements contained consensus binding sites for C/EBP transcription factors. Then, we hypothesized that C/EBPβ binds to these sites, activates transcription of Csf1r gene and promotes survival of Ly6C^– monocytes. To evaluate this hypothesis, we utilized an expression vector, in which green fluorescent protein (GFP) is driven by a combination of the Csf1r promoter and FIRE sequences (Csf1r-EGFP-FIRE) (a kind gift from Drs Clare P and David A Hume, University of Edinburgh). When a C/EBPβ expression vector was co-transfected with the vector containing Csf1r-EGFP-FIRE into HEK293 cells, the frequencies of GFP positive cells were significantly higher when compared to a control vector (C/EBPβ vs control; 4.6±0.6 vs 1.6±1.0, p=0.01), suggesting that C/EBPβ regulates Csf1rexpression through these elements. We are currently evaluating the significance of C/EBP consensus binding sites in the promoter and the enhancer. ChIP PCR is also in progress to further verify our hypothesis.

Collectively, these results suggest that Csf1r is a critical downstream target of C/EBPβ in Ly6C^– monocytes.