The function of the NF-E2 transcription factor, a p45/small Maf heterodimer, was analyzed in the erythroleukemia cell lines MEL and CB3. In contrast to MEL cells, CB3 cells are null for p45 and thus express only extremely low levels of adult globin transcripts upon induction by agents promoting erythroid differentiation. We investigated the response of erythroleukemia cells to hemin treatment. Hemin rapidly induces beta-globin gene transcript levels in MEL cells, but not in CB3 cells. Stable expression of the large p45 NF-E2 subunit in CB3 cells restores hemin mediated beta-globin gene transcription, suggesting that the presence of a functional NF-E2 is required for strong induction of beta-globin mRNA levels by hemin in erythroleukemia cells. We performed mutagenesis of two potential heme-regulatory motifs (HRMs) in p45 NF-E2 and found that the mutated versions are expressed and can still recognize a NF-E2 DNA binding element. In addition, we showed that p45 NF-E2 HRM mutants are able to restore beta-globin gene transcription in CB3 cells upon induction by hemin. Our results suggest that globin gene activation by heme appears to be independent of the putative HRMs in the p45 subunit of the NF-E2 transcription factor.
Murine erythroleukemia (MEL) cells provide a valuable model to study the molecular events leading to erythroid differentiation. Maturing erythroid cells synthesize large quantities of hemoglobin, a process requiring the coordinated synthesis of heme and globin. Here, we investigated the role of the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways in the differentiation of MEL cells. We determined the effect of the MEK1/2 inhibitor U0126 that blocks the ERK1/2 pathway, and the p38 inhibitor SB202190 on the differentiation potential of MEL cells induced by hexamethylene bisacetamide (HMBA). We found that treatment of HMBA induced MEL cells with the ERK1/2 pathway inhibitor U0126 results in higher hemoglobin levels. Using a fluorometric assay, we determined that intracellular heme levels also increased. Immunoblot studies showed an increase in globin protein levels. In contrast, treatment of MEL cells with the p38 inhibitor SB202190 has the opposite effect, leading to decreased amounts of heme and hemoglobin. In addition, inhibition of the p38 pathways results in lower transferrin receptor levels. Our results suggest that the ERK1/2 and p38 pathways play antagonistic roles in HMBA induced erythroid differentiation in MEL cells. This data also provides a novel link between MAPK signaling and the regulation of heme biosynthesis and iron uptake in erythroid cells.
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