Differential Impact of Transcriptional vs. Post-Transcriptional Control Mechanisms in the Regulation of Transferrin Receptor-1 and -2 in Human Myeloid Cells.

Abstract

Iron homeostasis is critically modulated by the expression of transferrin receptors (TfR)-1 and 2 and ferritin. In most tissues TfR1 and ferritin expression is controlled by iron availability at the post-transcriptional level. Furthermore, TfR expression is related to the proliferative state of the cells as well as the induction of differentiation. The available data on regulation of iron metabolism in human myeloid cells are limited and conflicting. In the present study, we evaluated TfR1/TfR2 expression in: (i) human acute myeloid leukemia (AML) cell lines (MOLM-16, HL-60, NB4, OCI-AML, ME1, MV4-11, NOMO-1, OCI-M1, MO7e, corresponding to FAB-M0 through FAB-M7 subtypes, respectively); (ii) a model system of human erythropoiesis, in which peripheral blood (PB) CD34+ cells from normal individuals are cultured in serum-free StemSpan medium in the presence of stem-cell factor (100ng/ml) and erythropoietin (4u/ml). Real-time RT-PCR was used to quantitate TfR1 and TfR2 (alpha/beta isoforms) mRNA levels. Western blotting experiments for TfR1/2 were performed with appropriate antibodies on cell lysates. TfR1 mRNA levels were divergent in AML cell lines; the highest expression was observed in MOLM-16 cells. TfR2 mRNA levels (both α and β isoforms) were low. TfR1 and TfR2 proteins were detected in all cell lines; TfR1 levels were uniform, whereas TfR2 protein expression varied significantly (highest levels in MOLM-16 cells). In the PB-CD34+ model system of erythropoiesis, TfR1 mRNA levels increased significantly during cell proliferation and erythroid maturation (4–7-fold over days 3–12). In contrast, TfR2α mRNA transcripts were low at the proerythroblast stage, then increased ×2–4-fold over days 4–12 and thereafter declined ~2.5 fold up to terminal differentiation. TfR2β mRNA levels were also low in days 1–3 of culture and significantly declined throughout erythroid differentiation. TfR1 protein was detected from day 3; thereafter, a significant increase was observed during erythroid maturation (days 3–9), whereas a gradual decrease was observed after day 12. In contrast, TfR2 protein was undetectable at day 3; similar to TfR-1, TfR-2 levels peaked on day 9 and then gradually declined. TfR1 and -2 mRNA stability was analyzed with the addition of actinomycin-D (actD: 5μg/ml) for 24 and 48hrs on day 7 of culture (proerythroblast stage). The levels of both TfR1 and TfR2α/β mRNA transcripts increased by 1.4–3.0-fold in actD-treated-CD34+ cells. These findings suggest that: (i) TfR1 is highly expressed from early erythroid stages without significant changes throughout erythroid maturation, in a context of progressively increasing transcription as well as prominent post-transcriptional regulation, (ii) TfR2 mRNA isoforms are differentially expressed throughout erythropoiesis (iii) AML cell lines show uniformly high TfR1 but variable TfR2 protein expression, suggesting that TfR2 might have only a subsidiary role in iron metabolism in malignant myeloid cells.