Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP epsilon).
Human C/EBP epsilon 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/EBP epsilon 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/EBP epsilon mRNA occurred as early as 1 hour after exposure of NB4 cells to 9-cis RA (5 x 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/EBP epsilon 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/EBP epsilon mRNA and an increase in granulocytic differentiation, but exposure to either DMSO or HMBA alone had no effect on base levels of C/EBP epsilon and did not induce differentiation. Macrophage-differentiation of NB4 reduced levels of C/EBP epsilon mRNA. Nuclear run-off assays and half-life studies showed that accumulation of C/EBP epsilon mRNA by 9-cis RA was due to enhanced transcription. Furthermore, this C/EBP epsilon mRNA accumulation did not require synthesis of new protein factors because 9-cis RA induced C/EBP epsilon mRNA accumulation in the absence of new protein synthesis. ATRA also induced expression of C/EBP epsilon protein in NB4 cells, as shown by Western blotting. In contrast to the increase of C/EBP epsilon 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/EBP epsilon mRNA levels. In summary, we have discovered that expression of C/EBP epsilon mRNA is markedly enhanced as the NB4 promyelocytes are induced by retinoids to differentiate towards granulocytes. This induction of C/EBP epsilon mRNA expression is transcriptionally mediated and occurs in the absence of synthesis of additional protein factors. We suspect that the C/EBP epsilon promoter/enhancer contains a retinoic acid-response element that is directly stimulated by retinoids.
Chih
D Y
DY
Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048, USA.
Chumakov
A M
AM
Park
D J
DJ
Silla
A G
AG
Koeffler
H P
HP
D015152:Blotting, Northern; D022762:CCAAT-Enhancer-Binding Proteins; D002454:Cell Differentiation; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D006098:Granulocytes / Q000166:cytology; D018922:HL-60 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors