Modulation of mRNA Expression of a Novel Human Myeloid-Selective CCAAT/Enhancer Binding Protein Gene (C/EBPε)

Abstract

Human C/EBPε is a newly cloned gene coding for a CCAAT/enhancer binding protein that may be involved in the regulation of myeloid differentiation. Our studies showed that levels of C/EBPε mRNA were markedly increased in NB4 cells (promyelocytic leukemia line), because they were induced by 9-cis retinoic acid (9-cis RA) to differentiate towards granulocytes. Accumulation of C/EBPε mRNA occurred as early as 1 hour after exposure of NB4 cells to 9-cis RA (5 × 10−7 mol/L); and at 48 hours, levels were increased by 5.1-fold. Dose-response studies showed that 10−7 to 10−6 mol/L 9-cis RA (12 hours) resulted in peak levels of C/EBPε mRNA; but even 10−10 mol/L 9-cis RA increased levels of these transcripts. NB4 cells pulse-exposed (30 minutes) to all-trans retinoic acid (ATRA), washed, and cultured (3 days) with either dimethylsulfoxide (DMSO) or hexamethylene bisacetamide (HMBA) had a prominent increase in levels of C/EBPε mRNA and an increase in granulocytic differentiation, but exposure to either DMSO or HMBA alone had no effect on base levels of C/EBPε and did not induce differentiation. Macrophage-differentiation of NB4 reduced levels of C/EBPε mRNA. Nuclear run-off assays and half-life studies showed that accumulation of C/EBPε mRNA by 9-cis RA was due to enhanced transcription. Furthermore, this C/EBPε mRNA accumulation did not require synthesis of new protein factors because 9-cis RA induced C/EBPε mRNA accumulation in the absence of new protein synthesis. ATRA also induced expression of C/EBPε protein in NB4 cells, as shown by Western blotting. In contrast to the increase of C/EBPε in 9-cis RA–mediated granulocytic differentiation, the DMSO-induced differentiation of HL-60 cells down the granulocytic pathway was associated with an initial reduction of C/EBPε mRNA levels. In summary, we have discovered that expression of C/EBPε mRNA is markedly enhanced as the NB4 promyelocytes are induced by retinoids to differentiate towards granulocytes. This induction of C/EBPε mRNA expression is transcriptionally mediated and occurs in the absence of synthesis of additional protein factors. We suspect that the C/EBPε promoter/enhancer contains a retinoic acid-response element that is directly stimulated by retinoids.

All-trans retinoic acid-induced expression of bactericidal/permeability-increasing protein (BPI) in human myeloid cells correlates to binding of C/EBPβ and C/EBPε to the BPI promoter

“…C/EBPε is involved in transcriptional regulation of genes encoding specific granule proteins such as LF and collagenase [43,44], and C/EBPε is essential for late granulocytic differentiation [45]. Our present results are consistent with previous reports that C/EBPε is up-regulated in NB4 after treatment with ATRA via RAREs in the C/EBPε promoter [32,46] and that C/EBP␤ expression and DNA binding are induced rapidly by ATRA via a PM leukemia-retinoic acid receptor ␣-dependent pathway in NB4 cells [33].…”

The IFN-inducible nucleoprotein IFI 16 is expressed in cells of the monocyte lineage, but is rapidly and markedly down-regulated in other myeloid precursor populations

“…For example, mice genetically engineered to lack expression of C/EBP-␤ show defects of macrophage function [37], whereas others lacking C/EBP-␣ expression have defective granulocyte development [38]. More recently, the expression of a novel member of this family (C/EBP-⑀) was shown to be inducible in NB4 and other myeloid cells, concomitant with differentiation to the granulocyte lineage [39]. The family of molecules represented by p202 in the mouse and by IFI 16 and MNDA in humans are also candidate molecules for such a function.…”

Nuclear Hormone Receptors Enable Macrophages and Dendritic Cells to Sense Their Lipid Environment and Shape Their Immune Response

“…However, the contribution of RAR to early myeloid development and to the granulocyte lineage is more prominent than to the monocytic development (see below). RAR␣ induces C/EBP⑀ (87,399,575), CD18 (12, 63), CD38 (128), RTP801, a recently discovered inhibitor of the mTOR pathway (155), interferon regulatory factor-1 (IRF1) (327), and leukocyte alkaline phosphatase (158) and mediates cell cycle arrest, a key phenomenon during differentiation (477,545). M-CSF, M-CSF receptor, G-CSF, GM-CSF, and GM-CSF receptor were also reported to be induced by retinoic acid in bone marrow stromal cells (111,216,373,552).…”