Modulation of Iron Homeostasis during Erythroid Differentiation Is Mediated by Complementary Transcriptional and Post-Transcriptional Mechanisms.

Iron is essential for cell proliferation, heme synthesis and a variety of cellular metabolic processes. Transferrin receptors (TfR)-1 and -2, ferritin and DMT1/Nramp2 are critically implicated in iron homeostasis. In this study, we evaluated TfR1/TfR2 (both alpha and beta isoforms), ferritin H-/-L-subunit and DMT1/Nramp2 mRNA expression as well as TfR1 protein expression in a model system of human erythropoiesis in an attempt to understand mechanisms critically involved in iron metabolism during normal erythroid maturation. CD34+ erythroid precursors collected from peripheral blood samples of normal individuals were cultured in free serum StemSpan medium in the presence of stem-cell factor (SCF, 100ng/ml) and erythropoietin (4u/ml). Real-time quantitative RT-PCR analysis showed that TfR1 mRNA levels increased significantly during cell proliferation and erythroid maturation (4–7-fold over days 4–12); in contrast, TfR2-alpha mRNA transcripts were low at the proerythroblast stage, then showed a slight increase (1.2–1.3-fold over days 4–12) and eventually remained stable up to terminal differentiation. TfR2-beta mRNA levels were generally low and did not fluctuate significantly throughout erythroid differentiation. TfR1 and TfR2-alpha mRNA stability was analyzed in cultures exposed to actinomycin-D (actD: 5μg/ml) for 24 and 48hrs on day 8 of culture (proerythroblast stage). The levels of both TfR1 and TfR2-alpha mRNA transcripts increased by 2.0–3.0-fold in actD-treated-CD34+ cells. Western blotting experiments were performed with an anti-TfR1-specific antibody on lysates derived from CD34+ cells throughout erythroid differentiation (days 3–14 of culture); in all experiments, actin served as control. Quantitative gel-banding densitometry was performed using Epson GT-8000 Laser Scanner. A significant increase of TfR1 protein was observed during erythroid maturation (from day 3 up to day 9), whereas a gradual decrease was observed after day 12 of culture. Assessment of H- and L-ferritin mRNA content at different stages of erythroid maturation demonstrated slightly higher levels of H-ferritin, albeit without significant changes throughout erythroid maturation. Similar results were obtained for DMT-1/Nramp-2 mRNA. These findings suggest that:

1. TfR1 upregulation starting from early erythroid stages and ongoing throughout erythroid maturation is mediated via transcriptional and post-transcriptional mechanisms, especially at more advanced differentiation stages;

2. TfR2-alpha expression is uniformly low throughout erythroid differentiation and regulated by both transcriptional and post-transcriptional mechanisms.

These observations suggest that increased iron demands of developing erythroid cells might be met by increased TfR-1 expression, in a context of high TfR-1 mRNA transcripts with increased stabiliy at more advanced maturation stages.